Methods and compositions comprising DNA damaging agents and p53

ABSTRACT

The present invention relates to the use of tumor suppressor genes in combination with a DNA damaging agent or factor for use in killing cells, and in particular cancerous cells. A tumor suppressor gene, p53, was delivered via a recombinant adenovirus-mediated gene transfer both in vitro and in vivo, in combination with a chemotherapeutic agent. Treated cells underwent apoptosis with specific DNA fragmentation. Direct injection of the p53-adenovirus construct into tumors subcutaneously, followed by intraperitoneal administration of a DNA damaging agent, cisplatin, induced massive apoptotic destruction of the tumors. The invention also provides for the clinical application of a regimen combining gene replacement using replication-deficient wild-type p53 adenovirus and DNA-damaging drugs for treatment of human cancer.

The present application is a continuation-in-part of co-pending U.S.patent application Ser. No. 08/145,826, filed Oct. 29, 1993; which is acontinuation-in-part of U.S. patent application Ser. No. 07/960,543,filed Oct. 13, 1992; which is a continuation-in-part of U.S. Ser. No.07/665,538, filed Mar. 6, 1991; the entire text and figures of whichdisclosures are incorporated herein by reference without disclaimer.

The government owns rights in the present invention pursuant to NIHgrants ROI CA 45187 and CA 16672, and Training Grants CA 09611 and CA45225.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the area of novel strategiesfor the improvement of chemotherapeutic intervention. In other aspects,the present invention provides novel methods and compositions thatcombine the potency of DNA damaging agents with the combined delivery ofa tumor suppressor. The combination of DNA damaging factors with theheterologous expression of a tumor suppressor gene lead to a pronouncedsynergy over and above the actions of the individual components.

2. Description of Related Art

Current treatment methods for cancer, including radiation therapy,surgery, and chemotherapy, are known to have limited effectiveness. Lungcancer alone kills more than 140,000 people annually in the UnitedStates. Recently, age-adjusted mortality from lung cancer has surpassedthat from breast cancer in women. Although implementation ofsmoking-reduction programs has decreased the prevalence of smoking, lungcancer mortality rates will remain high well into the 21st century. Therational development of new therapies for lung cancer will depend on anunderstanding of the biology of lung cancer at the molecular level.

It is now well established that a variety of cancers are caused, atleast in part, by genetic abnormalities that result in either the overexpression of one or more genes, or the expression of an abnormal ormutant gene or genes. For example, in many cases, the expression ofoncogenes is known to result in the development of cancer. “Oncogenes”are genetically altered genes whose mutated expression product somehowdisrupts normal cellular function or control (Spandidos et al., 1989).

Most oncogenes studied to date have been found to be “activated” as theresult of a mutation, often a point mutation, in the coding region of anormal cellular gene, i.e., a “proto-oncogene”, that results in aminoacid substitutions in the expressed protein product. This alteredexpression product exhibits an abnormal biological function that takespart in the neoplastic process (Travali et al., 1990). The underlyingmutations can arise by various means, such as by chemical mutagenesis orionizing radiation. A number of oncogenes and oncogene families,including ras, myc, neu, raf, erb, src, fms, jun and abl, have now beenidentified and characterized to varying degrees (Travali et al., 1990;Bishop, 1987).

During normal cell growth, it is thought that growth-promotingproto-oncogenes are counterbalanced by growth-constraining tumorsuppressor genes. Several factors may contribute to an imbalance inthese two forces, leading to the neoplastic state. One such factor ismutations in tumor suppressor genes (Weinberg, 1991).

An important tumor suppressor gene is the gene encoding the cellularprotein, p53, which is a 53 kD nuclear phosphoprotein that controls cellproliferation. Mutations to the p53 gene and allele loss on chromosome17p, where this gene is located, are among the most frequent alterationsidentified in human malignancies. The p53 protein is highly conservedthrough evolution and is expressed in most normal tissues. Wild-type p53has been shown to be involved in control of the cell cycle (Mercer,1992), transcriptional regulation (Fields et al., 1990, and Mietz etal., 1992), DNA replication (Wilcock and Lane, 1991, and Bargonetti etal., 1991), and induction of apoptosis (Yonish-Rouach et al., 1991, and,Shaw et al., 1992).

Various mutant p53 alleles are known in which a single base substitutionresults in the synthesis of proteins that have quite different growthregulatory properties and, ultimately, lead to malignancies (Hollsteinet al., 1991). In fact, the p53 gene has been found to be the mostfrequently mutated gene in common human cancers (Hollstein et al., 1991;Weinberg, 1991), and is particularly associated with those cancerslinked to cigarette smoke (Hollstein et al., 1991; Zakut-Houri et al.,1985). The overexpression of p53 in breast tumors has also beendocumented (Casey et al., 1991).

One of the most challenging aspects of gene therapy for cancer relatesto utilization of tumor suppressor genes, such as p53. It has beenreported that transfection of wild-type p53 into certain types of breastand lung cancer cells can restore growth suppression control in celllines (Casey et al., 1991; Takahasi et al., 1992). Although DNAtransfection is not a viable means for introducing DNA into patients'cells, these results serve to demonstrate that supplying wild type p53to cancer cells having a mutated p53 gene may be an effective treatmentmethod if an improved means for delivering the p53 gene could bedeveloped.

Gene delivery systems applicable to gene therapy for tumor suppressionare currently being investigated and developed. Virus-based genetransfer vehicles are of particular interest because of the efficiencyof viruses in infecting actual living cells, a process in which theviral genetic material itself is transferred. Some progress has beenmade in this regard as, for example, in the generation of retroviralvectors engineered to deliver a variety of genes. However, majorproblems are associated with using retroviral vectors for gene therapysince their infectivity depends on the availability of retroviralreceptors on the target cells, they are difficult to concentrate andpurify, and they only integrate efficiently into replicating cells.

Tumor cell resistance to chemotherapeutic drugs represents a majorproblem in clinical oncology. NSCLC accounts for at least 80% of thecases of lung cancer; patients with NSCLC are, however, generallyunresponsive to chemotherapy (Doyle, 1993). One goal of current cancerresearch is to find ways to improve the efficacy of gene replacementtherapy for cancer by investigating interaction between the gene productand chemotherapeutic drugs. The herpes simplex-thymidine kinase (HS-tK)gene, when delivered to brain tumors by a retroviral vector system,successfully induced susceptibility to the antiviral agent ganciclovir(Culver, et al., 1992). The HS-tK gene product is an exogenous viralenzyme, whereas the wt-p53 protein is expressed in normal tissues,suggesting that the modulation of chemoresistance by alterations inwt-p53 expression might be an alternative approach using a pathwaymediated by an endogenous genetic program.

An adenovirus system has potential advantages for gene delivery in vivo,such as ease of producing high titer virus, high infection efficiency,and infectivity for many types of cells. The stability and duration ofexpression of the introduced gene are still controversial, however. Theincrease in p53 levels in cells that are sensitive to chemotherapeuticdrugs can occur within 6 hours after DNA-damaging stimuli (Fritsche, etal., 1993, Zhan, et al., 1993), although increased p53 DNA bindingactivity can be reversed over the course of 4 hours if the stimulus isremoved (Tishler, et al., 1993). Therefore, a high level of p53expression can be maintained even after cessation of drug exposure. Theexpression of wt-p53 protein by Ad-p53 peaks at postinfection day 3(14-fold greater than endogenous wild type) and decreases to a low levelby day 9 (Zhang, et al., 1993). This suggests that a transiently highlevel of wt-p53 expression is sufficient to initiate the cytotoxicprogram in the cancer cell.

p53 has an important role as a determinant of chemosensitivity in humanlung cancer cells. A variety of treatment protocols, including surgery,chemotherapy, and radiotherapy, have been tried for human NSCLC, but thelong-term survival rate remains unsatisfactory. What is needed is acombination therapy that is used alone or as an effective adjuvanttreatment to prevent local recurrence following primary tumor resectionor as a treatment that could be given by intralesional injections indrug-resistant primary, metastatic, or locally recurrent lung cancer.Compositions and methods are also needed to developed, explore andimprove clinical applicability of novel compositions for the treatmentof cancer. Furthermore these methods and compositions must prove theirvalue in an in vivo setting.

SUMMARY OF THE INVENTION

The present invention addresses the need for improved therapeuticpreparations for use in killing cells by combining the effects of atumor suppressor gene or protein and a DNA damaging agent or factor. Thepresent invention also provides compositions and methods, includingthose that use viral mediated gene transfer, to promote expression of awild-type tumor suppressor gene, such as p53, in target cells and todeliver an agent or factor that induces DNA damage. The inventorssurprisingly found that using the compositions disclosed herein, theywere able to induce programmed cell death, also known as apoptosis, in avery significant number of target cells.

Using the present invention the inventors have demonstrated a remarkableeffect in controlling cell growth and in particular, tumor cell growth.Tumor cell formation and growth, also known as “transformation”,describes the formation and proliferation of cells that have lost theirability to control cellular division, that is, they are cancerous. It isenvisioned that a number of different types of transformed cells arepotential targets for the methods and compositions of the presentinvention, such as: sarcomas, melanomas, lymphomas, and a wide varietyof solid tumors and the like. Although any tissue having malignant cellgrowth may be a target, lung and breast tissue are preferred targets.The present inventors disclose herein that a p53-expressing recombinantdelivery vector was able to markedly reduce the growth rate of cellswhen used in conjunction with a DNA damaging agent.

The invention provides, in certain embodiments, methods and compositionsfor killing a cell or cells, such as a malignant cell or cells, bycontacting or exposing a cell or population of cells with a p53 proteinor gene and one or more DNA damaging agents in a combined amounteffective to kill the cell(s). Cells that may be killed using theinvention include, e.g., undesirable but benign cells, such as benignprostate hyperplasia cells or over-active thyroid cells; cells relatingto autoimmune diseases, such as B cells that produce antibodies involvedin arthritis, lupus, myasthenia gravis, squamous metaplasia, dysplasiaand the like. Although generally applicable to killing all undesirablecells, the invention has a particular utility in killing malignantcells. “Malignant cells” are defined as cells that have lost the abilityto control the cell division cycle, as leads to a “transformed” or“cancerous” phenotype.

To kill cells, such as malignant or metastatic cells, using the methodsand compositions of the present invention, one would generally contact a“target” cell with a p53 protein or gene and at least one DNA damagingagent in a combined amount effective to kill the cell. This process mayinvolve contacting the cells with the p53 protein or gene and the DNAdamaging agent(s) or factor(s) at the same time. This may be achieved bycontacting the cell with a single composition or pharmacologicalformulation that includes both agents, or by contacting the cell withtwo distinct compositions or formulations, at the same time, wherein onecomposition includes the p53 protein or gene and the other includes theDNA damaging agent.

Naturally, it is also envisioned that the target cell may be firstexposed to the DNA damaging agent(s) and then contacted with a p53protein or gene, or vice versa. However, in embodiments where the DNAdamaging factor and p53 are applied separately to the cell, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the DNA damaging agent andp53 would still be able to exert an advantageously combined effect onthe cell. In such instances, it is contemplated that one would contactthe cell with both agents within about 12-24 hours of each other, andmore preferably within about 6-12 hours of each other, with a delay timeof only about 12 hours being most preferred.

The terms “contacted” and “exposed”, when applied to a cell, are usedherein to describe the process by which a tumor suppressor gene orprotein, such as p53, and a DNA damaging agent or factor are deliveredto a target cell or are placed in direct juxtaposition with the targetcell. To achieve cell killing, both agents are delivered to a cell in acombined amount effective to kill the cell, i.e., to induce programmedcell death or apoptosis. The terms, “killing”, “programmed cell death”and “apoptosis” are used interchangeably in the present text to describea series of intracellular events that lead to target cell death. Theprocess of cell death involves the activation of intracellular proteasesand nucleases that lead to, for example, cell nucleus involution andnuclear DNA fragmentation. An understanding of the precise mechanisms bywhich various intracellular molecules interact to achieve cell death isnot necessary for practicing the present invention.

DNA damaging agents or factors are defined herein as any chemicalcompound or treatment method that induces DNA damage when applied to acell. Such agents and factors include radiation and waves that induceDNA damage, such as, γ-irradiation, X-rays, UV-irradiation, microwaves,electronic emissions, and the like. A variety of chemical compounds,also described as “chemotherapeutic agents”, function to induce DNAdamage, all of which are intended to be of use in the combined treatmentmethods disclosed herein. Chemotherapeutic agents contemplated to be ofuse, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16),camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), and evenhydrogen peroxide. The invention also encompasses the use of acombination of one or more DNA damaging agents, whether radiation-basedor actual compounds, such as the use of X-rays with cisplatin or the useof cisplatin with etoposide. In certain embodiments, the use ofcisplatin in combination with a p53 protein or gene is particularlypreferred as this compound.

Any method may also be used to contact a cell with a p53 protein, solong as the method results in increased levels of functional p53 proteinwithin the cell. This includes both the direct delivery of a p53 proteinto the cell and the delivery of a gene or DNA segment that encodes p53,which gene will direct the expression and production of p53 within thecell. In that protein delivery is subject to such drawbacks as proteindegradation and low cellular uptake, it is contemplated that the use ofa recombinant vector that expresses a p53 protein will provideparticular advantages.

A wide variety of recombinant plasmids and vectors may be engineered toexpresses a p53 protein and so used to deliver p53 to a cell. Theseinclude, for example, the use of naked DNA and p53 plasmids to directlytransfer genetic material into a cell (Wolfe et al., 1990); formulationsof p53-encoding DNA trapped in liposomes (Ledley et al., 1987) or inproteoliposomes that contain viral envelope receptor proteins (Nicolauet al., 1983); and p53-encoding DNA coupled to a polylysine-glycoproteincarrier complex.

The use of recombinant viruses engineered to express p53 is alsoenvisioned. A variety of viral vectors, such as retroviral vectors,herpes simplex virus (U.S. Pat. No. 5,288,641, incorporated herein byreference), cytomegalovirus, and the like may be employed, as describedby Miller (Miller, 1992); as may recombinant adeno-associated virus (AAVvectors), such as those described by U.S. Pat. No. 5,139,941,incorporated herein by reference; and, particularly, recombinantadenoviral vectors. Techniques for preparing replication-defectiveinfective viruses are well known in the art, as exemplified byGhosh-Choudhury & Graham (1987); McGrory et al. (1988); and Gluzman etal. (1982), each incorporated herein by reference.

To kill a cell in accordance with the present invention, one wouldgenerally contact the cell with a p53 protein or gene and a DNA damagingagent in a combined amount effective to kill the cell. The term “in acombined amount effective to kill the cell” means that the amount of p53and DNA damaging agents are sufficient so that, when combined within thecell, the cell is induced to undergo apoptosis. Although not required inall embodiments, the combined effective amount of p53 and DNA damagingagent will preferably be an amount that induces significantly more celldeath than the use of either element alone, and most preferably, thecombined effective amount will be an amount that induces synergisticcell death in comparison to the effects observed using either elementalone.

A number of in vitro parameters may be used to determine the effectproduced by the compositions and methods of the present invention. Theseparameters include, for example, the observation of net cell numbersbefore and after exposure to the compositions described herein, as wellas the size of multicellular tumor spheroids formed, such as thosecolonies formed in tissue culture. In vitro cell killing is particularlyshown in Example 7 of the present disclosure. Alternatively, one maymeasure parameters that are indicative of a cell that is undergoingprogrammed cell death, such as, the fragmentation of cellular genomicDNA into nucleosome size fragments, generally identified by separatingthe fragments by agarose gel electrophoresis, staining the DNA, andcomparing the DNA to a DNA size ladder. Nucleosome size fragments areidentified as a progressive steps or ladders of monomers and multimershaving a base unit of about 200 basepairs.

Similarly, a “therapeutically effective amount” is an amount of a p53protein or gene and DNA damaging agent that, when administered to ananimal in combination is effective to kill cells within the animal. Thisis particularly evidenced by the killing of cancer cells, such as lung,breast or colon cancer cells, within an animal or human subject that hasa tumor. “Therapeutically effective combinations” are thus generallycombined amounts of p53 and DNA damaging agents that function to killmore cells than either element alone, and preferably, combined amountsthat bring about a synergistic reduction in tumor burden.

Studying certain in vivo and ex vivo parameters of cell death aretherefore also effective means by which to assess the effectiveness ofthe composition and methods of the invention. For example, observingeffects on the inhibition of tumorigenicity, as measured by TdTexpression of frozen tissue sections or by using other staining methodsand target antigens, as known to skilled pathologists. Naturally, othermeans of determining tumor mass, growth, and viability may also be usedto assess the killing of target cells. In particular, one may assess theeffects in various animal model systems of cancer, including those inwhich human cancer cells are localized within the animal. Animal modelsof cancer, unlike those of AIDS, are known to be highly predictive ofhuman treatment regimens (Roth et al., editors (1989)). One exemplaryembodiment of a predictive animal model is that in which humansmall-cell lung cancer cells (H358 cells) are grown subcutaneously.Using this system, the inventors have shown that p53-bearing adenovirusinstilled intratumorally, along with the co-administration of achemotherapeutic agent, gives rise to a surprisingly effective tumorreduction.

A particularly preferred method of delivering a p53 protein to a cell isto contact the cell with a recombinant adenovirus virion or particlethat includes a recombinant adenoviral vector comprising a p53expression region positioned under the control of a promoter capable ofdirecting the expression of p53 in the given cell type.

The p53 expression region in the vector may comprise a genomic sequence,but for simplicity, it is contemplated that one will generally prefer toemploy a p53 cDNA sequence as these are readily available in the art andmore easily manipulated. In addition to comprising a p53 expression unitand a promoter region, the vector will also generally comprise apolyadenylation signal, such as an SV40 early gene, or protamine gene,polyadenylation signal, or the like.

In preferred embodiments, it is contemplated that one will desire toposition the p53 expression region under the control of a strongconstitutive promoter such as a CMV promoter, viral LTR, RSV, or SV40promoter, or a promoter associated with genes that are expressed at highlevels in mammalian cells such as elongation factor-1 or actinpromoters. All such variants are envisioned to be useful with thepresent invention. Currently, a particularly preferred promoter is thecytomegalovirus (CMV) IE promoter.

The p53 gene or cDNA may be introduced into a recombinant adenovirus inaccordance with the invention simply by inserting or adding the p53coding sequence into a viral genome. However, the preferred adenoviruseswill be replication defective viruses in which a viral gene essentialfor replication and/or packaging has been deleted from the adenoviralvector construct, allowing the p53 expression region to be introduced inits place. Any gene, whether essential (e.g., E1A, E1B, E2 and E4) ornon-essential (e.g., E3) for replication, may be deleted and replacedwith p53. Particularly preferred are those vectors and virions in whichthe E1A and E1B regions of the adenovirus vector have been deleted andthe p53 expression region introduced in their place, as exemplified bythe genome structure of FIG. 1.

Techniques for preparing replication defective adenoviruses are wellknown in the art, as exemplified by Ghosh-Choudhury and Graham (1987);McGrory et al. (1988); and Gluzman et al., each incorporated herein byreference. It is also well known that various cell lines may be used topropagate recombinant adenoviruses, so long as they complement anyreplication defect which may be present. A preferred cell line is thehuman 293 cell line, but any other cell line that is permissive forreplication, i.e., in the preferred case, which expresses E1A and E1Bmay be employed. Further, the cells can be propagated either on plasticdishes or in suspension culture, in order to obtain virus stocksthereof.

The invention is not limited to E1-lacking virus and E1-expressing cellsalone. Indeed, other complementary combinations of viruses and hostcells may be employed in connection with the present invention. Viruslacking functional E2 and E2-expressing cells may be used, as may viruslacking functional E4 and E4-expressing cells, and the like. Where agene which is not essential for replication is deleted and replaced,such as, for example, the E3 gene, this defect will not need to bespecifically complemented by the host cell.

Other than the requirement that the adenovirus vectors be engineered toexpress p53, the nature of the initial adenovirus is not believed to becrucial to the successful practice of the invention. The adenovirus maybe of any of the 42 different known serotypes or subgroups A-F.Adenovirus type 5 of subgroup C is the preferred starting material inorder to obtain the conditional replication-defective adenovirus vectorfor use in the method of the present invention. This is becauseAdenovirus type 5 is a human adenovirus about which there is significantamount of biochemical and genetic information known, and which hashistorically been used for most constructions employing adenovirus as avector.

The methods and compositions of the present invention are equallysuitable for killing a cell or cells both in vitro and in vivo. When thecells to be killed are located within an animal, e.g., lung, breast orcolon cancer cells or other cells bearing a p53 mutation, both the p53protein or gene and the DNA damaging agent will be administered to theanimal in a pharmacologically acceptable form. The term “apharmacologically acceptable form”, as used herein, refers to both theform of any composition that may be administered to an animal, and alsothe form of contacting an animal with radiation, i.e., the manner inwhich an area of the animals body is irradiated, e.g., withγ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions,and the like. The use of DNA damaging radiation and waves is known tothose skilled in the art of irradiation therapy.

The present invention also provides advantageous methods for treatingcancer that, generally, comprise administering to an animal or humanpatient with cancer a therapeutically effective combination of a p53protein or gene and a DNA damaging agent. This may be achieved using arecombinant virus, particularly an adenovirus, that carries a vectorcapable of expressing p53 in the cells of the tumor. The p53 genedelivering composition would generally be administered to the animal,often in close contact to the tumor, in the form of a pharmaceuticallyacceptable composition. Direct intralesional injection of atherapeutically effective amount of a p53 gene, such as housed within arecombinant virus, into a tumor site is one preferred method. However,other parenteral routes of administration, such as intravenous,percutaneous, endoscopic, or subcutaneous injection are alsocontemplated.

In treating cancer according to the invention one would contact thetumor cells with a DNA damaging agent in addition to the p53 protein orgene. This may be achieved by irradiating the localized tumor site withDNA damaging radiation such as X-rays, UV-light, γ-rays or evenmicrowaves. Alternatively, the tumor cells may be contacted with the DNAdamaging agent by administering to the animal a therapeuticallyeffective amount of a pharmaceutical composition comprising a DNAdamaging compound, such as, adriamycin, 5-fluorouracil, etoposide,camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.The DNA damaging agent may be prepared and used as a combinedtherapeutic composition, or kit, by combining it with a p53 protein,gene or gene delivery system, as described above.

The surprising success of the present invention is evidenced by thefinding that using Ad5CMV-p53 virus in combination with cisplatinyielded profound results in studies using a nude mouse model. Thecombined virus-DNA damage therapy regimen significantly inhibited thetumorigenicity of H358 cells, a cell that normally produces asignificant tumor mass. The tumorigenicity of the lung cancer cells wasinhibited through the treatment by Ad5CMV-p53, but not by the controlvirus expressing luciferase, indicating that the p53 protein incombination with a DNA-damaging agent has great therapeutic efficacy.

A number of methods for delivering chemotherapeutic formulations,including DNA expression constructs, into eukaryotic cells are known tothose of skill in the art. In light of the present disclosure, theskilled artisan will be able to deliver both DNA damaging agents and p53proteins or genes to cells in many different effective ways.

For in vivo delivery of DNA, the inventors envision the use of any genedelivery system, such as viral- and liposome-mediated transfection. Asused herein, the term “transfection”, is used to describe the targeteddelivery of DNA to eukaryotic cells using delivery systems, such as,adenoviral, AAV, retroviral, or plasmid delivery gene transfer methods.The specificity of viral gene delivery may be selected to preferentiallydirect the gene to a particular target cell, such as by using virusesthat are able to infect particular cell types. Naturally, differentviral host ranges will dictate the virus chosen for gene transfer, aswell as the likely tumor suppressor gene to be expressed for killing agiven malignant cell type.

It is also envisioned that one may provide the DNA damagingchemotherapeutic agent through a variety of means, such as by usingparenteral delivery methods such as intravenous and subcutaneousinjection, and the like. Such methods are known to those of skill in theart of drug delivery, and are further described herein in the sectionsregarding pharmaceutical preparations and treatment.

For in vitro gene delivery, a variety of methods may be employed, suchas, e.g., calcium phosphate- or dextran sulfate-mediated transfection;electroporation; glass projectile targeting; and the like. These methodsare known to those of skill in the art, with the exact compositions andexecution being apparent in light of the present disclosure.

Other embodiments concern compositions, including pharmaceuticalformulations, comprising a p53 protein or gene in combination with a DNAdamaging agent, such as cisplatin. In such compositions, the p53 may bein the form a DNA segment, recombinant vector or recombinant virus thatis capable of expressing a p53 protein in an animal cell. Thesecompositions, including those comprising a recombinant viral genedelivery system, such as an adenovirus particle, may be formulated forin vivo administration by dispersion in a pharmacologically acceptablesolution or buffer. Preferred pharmacologically acceptable solutionsinclude neutral saline solutions buffered with phosphate, lactate, Tris,and the like.

Of course, in using viral delivery systems, one will desire to purifythe virion sufficiently to render it essentially free of undesirablecontaminants, such as defective interfering viral particles orendotoxins and other pyrogens such that it will not cause any untowardreactions in the cell, animal or individual receiving the vectorconstruct. A preferred means of purifying the vector involves the use ofbuoyant density gradients, such as cesium chloride gradientcentrifugation.

Preferred pharmaceutical compositions of the invention are those thatinclude, within a pharmacologically acceptable solution or buffer, a p53protein, or more preferably a p53 gene, in combination with achemotherapeutic DNA damaging agent. Exemplary chemotherapeutic agentsare adriamycin, 5-fluorouracil, camptothecin, actinomycin-D, hydrogenperoxide, mitomycin C, cisplatin (CDDP), and etoposide (VP-16), with theuse of cisplatin being particularly preferred.

Still further embodiments of the present invention are kits for use inkilling cells, such as malignant cells, as may be formulated intotherapeutic kits for use in cancer treatment. The kits of the inventionwill generally comprise, in suitable container means, a pharmaceuticalformulation of a recombinant vector that is capable of expressing a p53protein in an animal cell, and a pharmaceutical formulation of a DNAdamaging agent. The recombinant vectors and DNA damaging agents may bepresent within a single container, or these components may be providedin distinct or separate container means. In a preferred embodiment, therecombinant vector will be a recombinant p53-expressing adenoviralvector present within an adenovirus particle and the DNA damaging agentwill be cisplatin.

The components of the kit are preferably provided as a liquid solution,or as a dried powder. When the components are provided in a liquidsolution, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. When reagents orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Scheme for generation of recombinant p53 adenovirus. The p53expression cassette was inserted between the Xba I and Cla I sites ofpXCJL.1. The p53 expression vector (pEC53) and the recombinant plasmidpJM17 were cotransfected into 293 cells. The transfected cells weremaintained in medium until the onset of the cytopathic effect.Identification of newly generated p53 recombinant adenoviruses(Ad5CMV-p53) by PCR analysis of the DNA using DNA templates preparedfrom the CPE supernatants treated with Proteinase K and phenolextraction.

FIG. 2A. Map used for the structural analysis of Ad5CMV-p53 DNA. A mapof Ad5CMV-p53 genomic DNA, with the locations of the p53 expressioncassette, the PCR primers, and the restriction sites. The genome size isabout 35.4 kb, divided into 100 maps units (1 m.u.=0.35 kb). The p53expression cassette replaced the E1 region (1.3-9.2 m.u.) of the Ad5genome. Primer 1 is located in the first intron downstream of the humanCMV major IE gene promoter. Primer 2 is located in SV40 earlypolyadenylation signal. Both of the primers, 15-20 bp away from the p53cDNA insert at both ends, define a 1.40 kb PCR product. Primers 3 and 4are located at 11 m.u. and 13.4 m.u. of Ad5 genome, respectively, whichdefine a 0.86 kb viral-genome specific PCR product.

FIG. 2B. Agarose gel analysis of PCR products. Two pairs of primers thatdefine 1.4-kb (p53) and 0.86-kb (Ad5) DNA fragments were used in eachreaction. DNA templates used in each reaction were pEC53 plasmid (lane1), Ad5/RSV/GL2 DNA (lane 2), no DNA (lane 3), and Ad5CMV-p53 DNA (lane4). The lane labeled (M) corresponds to molecular weight markers.

FIG. 2C. Restriction mapping of Ad5CMV-p53 DNA. CsCl-gradient purifiedAd5CMV-p53 DNA samples were digested with no enzyme (U), Hind III (H),Bam HI (B), Eco RI (E), and Cla I (C), respectively, and analyzed on 1%agarose gel. The lanes labeled (M) are molecular weight markers.

FIGS. 3A, 3B, 3C and 3D. Observation of cytopathic effects on 293 byrecombinant adenovirus. FIGS. 3A, 3B, 3C and 3D are a series of phasecontrast images (×400) of 293 cells. FIGS. 3A, 3B, 3C and 3D are fourpanels of a single page figure. FIG. 2A, before transfection; FIG. 3B,negative control on day 12 posttransfection; FIG. 3C, onset of CPE onday 12 posttransfection; FIG. 3D, completion of CPE on day 14post-transfection.

FIGS. 4A, 4B, 4C, and 4D. Immunohistology of cells infected withrecombinant adenoviruses. FIGS. 4A, 4B, 4C and 4D are a series ofimmunohistological images of H358 cells. FIGS. 4A, 4B, 4C and 4D arefour panels of a single page figure. Infectivity of Ad5C MV-p53 in H358cells. H358 cells were infected with Ad5CMV-p53 or Ad5/RSV/GL2 at 50PFU/cell for 24 h. Medium alone was used as a mock infection. Theinfected cells were analyzed by immunostainings. FIG. 4A is a mockinfection probed with anti-p53 antibody. FIG. 4B are cells infected withthe Ad5/RSV/GL2 control and probed with anti-p53 antibody. FIG. 4C areAd5CMV-p53 infected cells probed with an unrelated antibody (MOPC-21).FIG. 4D are cells Ad5CMV-p53 infection probed with anti-p53 antibody.The anti-p53 antibody used was Pab 1801, and the avidin-biotin methodwas used for staining.

FIG. 5A. Coomassie-blue stained SDS-PAGE gel comparing the relativelevel of expression of exogenous p53 in H358 cells. H358 cell samplesthat were infected with Ad5CMV-p53 or Ad5/RSV/GL2 at 30 PFU/cell wereprepared 24 and 72 h after infection. Coomassie blue staining of anSDS-PAGE analysis, showing relative quantities of protein samplesloaded. Lanes 1 and 4 contain the samples of the Ad5/RSV/GL2-infectedcells. Lanes 2 and 3 contain the samples of the cells infected with twoindividual stocks of Ad5CMV-p53 at 24 h after infection. Lanes 5 and 6are the Ad5CMV-p53-infected cell samples collected at 72 h afterinfection. Lane 7 is mock-infected H358 sample 72 h after infection.Lane M, prestained molecular weight markers in kDa (GIBCO-BRL).

FIG. 5B. Western blot analysis of the identical lane setting gel as thatof the SDS-PAGE in FIG. 5A. The relative levels of expression of p53were analyzed by Western blotting-using anti-p53. Primary antibodiesused were monoclonal antibodies against p53 protein (PAb 1801, OncogeneScience Inc.) and β-actin (Amersham Inc.). The HRP-conjugated secondantibody and ECL developer were from Amershem Inc. viral-infected H358cells by Western Blotting. Western blot of FIG. 5B have an equivalentsetup and order to those in FIG. 5A.

FIG. 6. Time course of the p53 expression, determined by Westernblotting. Multiple dishes of H358 cells were infected with Ad5CMV-p53 at10 PFU/cell. Cell lysates were prepared at indicated time points afterinfection. Western blotting was probed with anti-p53 and anti-actinantibodies simultaneously. The lanes designated ‘C’ represent negativecontrols. The histogram represents the relative quantities of p53 asdetermined by a densitometer.

FIG. 7A. Growth curve of virally-infected human lung cancer cells ofcell lines H358. Cells were inoculated at 10⁵ cells per dish (60 mm) and6 dishes per cell line. After 24 hours, the cells were infected withAd5CMV-p53 or Ad5/RSV/GL2 at 10 m.o.i. (Multiplicity of infection, i.e.,PFU/cell). After infection cells were counted daily for 6 days. Thegrowth curves represent data obtained from 4 separate studies.

FIG. 7B. Growth curve of virally-infected human lung cancer cells ofcell line H322. Cells were inoculated at 10⁵ cells per dish (60 mm) and6 dishes per cell line. After 24 hours, the cells were infected withAd5CMV-p53 or Ad5/RSV/GL2 at 10 m.o.i. (Multiplicity of infection, i.e.,PFU/cell). After infection cells were counted daily for 6 days. Thegrowth curves represent data obtained from 4 separate studies.

FIG. 7C. Growth curve of virally-infected human lung cancer cells ofcell line H460. Cells were inoculated at 10⁵ cells per dish (60 mm) and6 dishes per cell line. After 24 hours, the cells were infected withAd5CMV-p53 or Ad5/RSV/GL2 at 10 m.o.i. (Multiplicity of infection, i.e.,PFU/cell). After infection cells were counted daily for 6 days. Thegrowth curves represent data obtained from 4 separate studies.

FIG. 8. Flow chart of tests of Ad5CMV-p53 in orthotopic lung cancermodel. The dosages and schedule of treatment of nude mice inoculatedwith H226Br cells and viruses are summarized in the flow chart.

FIGS. 9A, 9B, 9C, and 9D. Samples of the lung and mediastinum dissectionfrom treated and control mice. FIGS. 9A, 9B, 9C, and 9D are four panelsof a single FIGURE. The mice were sacrificed at the end of the 6-weekposttreatment period. The lung and mediastinum tissues were dissectedfor evaluation of tumor formation. FIG. 9A is a sample of mediastinalblock from a normal nude mice; FIG. 9B is the mediastinal block samplefrom the vehicle (PBS)-treated mice; FIG. 9C is the mediastinal blocksample from the Ad5CMV-p53-treated mice; FIG. 9D is the mediastinalblock sample from the Ad5/RSV/GL2-treated mice. Arrows indicate thetumor masses.

FIG. 10A. The effects of continuous exposure to CDDP on the growth ratesof parental, Ad-Luc-infected, and Ad-p53-infected H358 cells. H358 cells(1.5×10⁵ cells/well) were seeded in duplicate on a 24-well plate. After24 hours, 100 μl of medium, Ad-Luc viral stock (10⁸ PFU/ml), or Ad-p53viral stock (10⁸ PFU/ml) was added. Following an additional 24-hourincubation, the medium that contained virus was replaced with freshmedium that contained 10 μg/ml of CDDP.

FIG. 10B. 24-hour exposure to CDDP on the growth rates of parental,Ad-Luc-infected, and Ad-p53-infected H3.58 cells. Cells were exposed toCDDP (FIG. 10A) continuously or (FIG. 10B) for 24 hours followed byrecovery in drug-free medium. Cells that remained as an attachedmonolayer were assessed for viability over 5 days by measuring trypanblue uptake. The mean+/−SE is shown. The day 5 cell number for theAd-p53:CDDP group differs significantly from all other groups for both Aand B (p<0.05 by Student's t-test).

FIG. 10C. The effects of different concentrations of CDDP on theviability of Ad-p53-infected H358 cells. After 24-hour exposure to theAd-Luc or Ad-p53 virus, cells were treated with 0, 10, or 100 μg/ml ofCDDP for 24 hours and then assessed for viability.

FIG. 11A. Nucleosomal DNA fragmentation in Ad-p53-infected H358 cellsexposed to CDDP. Cells were infected and treated with CDDP for 24 hoursas described in the legend to FIG. 10.

FIGS. 11B, 11C, 11D, 11E and 11G. H358 cells that were grown on chamberslides, infected with Ad-p53 for 24 hours, treated with CDDP for anadditional 24 hours, and fixed for in situ labeling of DNAfragmentation. Pictured are parental H358 cells (B) without or (C) withCDDP; Ad-Luc-infected cells (D) without or (E) with CDDP; andAd-p53-infected cells (F) without or (G) with CDDP. The arrowhead showsan example of darkly stained nuclear fragments. Bar 100 μm.

FIG. 12A. Effect of the combination of Ad-p53 infection with CDDPtreatment on H358 tumor spheroids. Multicellular tumor spheroids of H358cells were prepared as previously described (Takahashi, et al. (1989)).On day 0, spheroids with a diameter of 150 to 200 μm were placed in a24-well agar coated plate and exposed to Ad-p53 or Ad-Luc for 24 hours.On day 1, medium with 10 μg/ml of CDDP was added following removal ofvirus-containing medium. On day 2, after a 24-hour incubation, theoverlay was replaced with 1 ml of fresh drug-free medium. Theperpendicular diameters were measured using an inverted microscope. Therelative volume change was calculated according to the formula a²×b/a₁²×b₁, where a and b are the smallest and largest diameters of thespheroid, respectively, and a₁ and b₁ are the diameters on day 1. Onlythe relative volume of the Ad-p53/CDDP spheroids is significantly less(p<0.05 by Student's t-test) than the control group (Ct1).

FIGS. 12B, 12C, 12D, and 12E. In situ dUTP labeling with TdT fordetection of apoptosis. H358 spheroids were fixed on day 3 and stainedas described in Materials and Methods of Example 7. (B) Controluntreated spheroid, (C) spheroid treated with CDDP, (D) Ad-p53-infectedspheroid, and (E) Ad-p53-infected spheroid treated with CDDP. Bar 10.0μm.

FIG. 13A. Induction of apoptosis by CDDP after in vivo infection withAd-p53 as measured by tumor volume changes. H358 cells (5×10⁶) in 0.1 mlHank's balanced salt solution were injected subcutaneously into theright flank of BALB/c female nu/nu mice. Thirty days later, 200 μl ofmedium alone or medium containing Ad-Luc (10⁸ PFU/ml) or Ad-p53 (10⁸PFU/ml) was injected into tumors with a diameter of 5 to 6 mm.Intratumoral injection (100 μl) and peritumoral injection in twoopposite sites (50 μl each) were performed. CDDP (3 mg/kg) or controlphysiological saline was given intraperitoneally. The tumors weremeasured with calipers in two perpendicular diameters without theknowledge of the treatment groups, and a tumor volume was calculated byassuming a spherical shape with the average tumor diameter calculated asthe square root of the product of cross-sectional diameters. Five micewere used for each treatment group and the mean+/−SE is shown. The datawas analyzed using the Student's t-test. The arrow shows the day oftreatment. Two independent determinations are shown. p<0.05 from day 5in test 1; p<0.05 from day 7 in test 2. (B-E)

FIGS. 13B, 13C, 13D, and 13E. Histologic study using the TdT-mediatedbiotin-dUTP labeling technique. Tumors were harvested 5 days after thebeginning of treatment and immediately embedded into O. C. T. compound.Frozen tissues were cut in a cryostat at 5-μm thicknesses. The sectionswere treated with 1 μg/ml proteinase K and stained as described in thelegend to FIG. 12. Pictured are H358 tumors treated with (B) CDDP alone,(C) Ad-p53 alone, or (D, E) Ad-p53 in the combination with CDDP.Bars=0.5 mm. All animal care was in accordance with the UT M.D. AndersonInstitutional Animal Care and Use Committee.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Molecular Events in Lung Cancer Development

Studies carried out by the present inventors has identified criticalmolecular events leading to the development and progression of cancer.This enabled the inventors to develop new methods for restoring certainnormal protein functions so that the malignant phenotype can besuppressed in vivo.

The most common lung cancer histologies (80%) are grouped under the termnon-small-cell lung cancer (NSCLC) and include squamous, adenocarcinoma,and large-cell undifferentiated. Many of the current data on themolecular biology of lung cancer come from the study of the moreuncommon small-cell lung cancer (SCLC). SCLC can be distinguished fromNSCLC by the neuroendocrine features of the cells; SCLC is veryresponsive to chemotherapy but recurs rapidly after treatment. NSCLCalso may serve as a model for other carcinogen-induced epithelialcancers. The approaches and observations developed in this study may beapplicable to other types of epithelial cancers.

Abundant evidence has accumulated that the process of malignanttransformation is mediated by a genetic paradigm. The major lesionsdetected in cancer cells occur in dominant oncogenes and tumorsuppressor genes. Dominant oncogenes have alterations in a class ofgenes called proto-oncogenes, which participate in critical normal cellfunctions, including signal transduction and transcription. Primarymodifications in the dominant oncogenes that confer the ability totransform include point mutations, translocations, rearrangements, andamplification. Tumor suppressor genes appear to require homozygous lossof function, by mutation, deletion, or a combination of these fortransformation to occur. Some tumor suppressor genes appear to play arole in the governance of proliferation by regulation of transcription.Modification of the expression of dominant and tumor suppressoroncogenes is likely to influence certain characteristics of cells thatcontribute to the malignant phenotype.

Despite increasing knowledge of the mechanisms involved inoncogene-mediated transformation, little progress has occurred indeveloping therapeutic strategies that specifically target oncogenes andtheir products. Initially, research in this area was focused on dominantoncogenes, as these were the first to be characterized. DNA-mediatedgene transfer studies showed acquisition of the malignant phenotype bynormal cells following the transfer of DNA from malignant human tumors.

B. p53 and p53 Mutations in Cancer

P53 is currently recognized as a tumor suppressor gene (Montenarh,1992). High levels have been found in many cells transformed by chemicalcarcinogenesis, ultraviolet radiation, and several viruses, includingSV40. The p53 gene is a frequent target of mutational inactivation in awide variety of human tumors and is already documented to be the mostfrequently-mutated gene in common human cancers (Mercer, 1992). It ismutated in over 50% of human NSCLC (Hollestein et al., 1991) and in awide spectrum of other tumors.

The p53 gene encodes a 375-amino-acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue.Interestingly, wild-type p53 appears to be important in regulating cellgrowth and division. Overexpression of wild-type p53 has been shown insome cases to be anti-proliferative in human tumor cell lines. Thus p53can act as a negative regulator of cell growth (Weinberg, 1991) and maydirectly suppress uncontrolled cell growth or indirectly activate genesthat suppress this growth. Thus, absence or inactivation of wild typep53 may contribute to transformation. However, some studies indicatethat the presence of mutant p53 may be necessary for full expression ofthe transforming potential of the gene.

Although wild-type p53 is recognized as a centrally important growthregulator in many cell types, its genetic and biochemical traits appearto have a role as well. Mis-sense mutations are common for the p53 geneand are essential for the transforming ability of the oncogene. A singlegenetic change prompted by point mutations can create carcinogenic p53.Unlike other oncogenes, however, p53 point mutations are known to occurin at least 30 distinct codons, often creating dominant alleles thatproduce shifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encodingwild-type p53 into two human breast cancer cell lines restores growthsuppression control in such cells (Casey et al., 1991). A similar effecthas also been demonstrated on transfection of wild-type, but not mutant,p53 into human lung cancer cell lines (Takahasi et al., 1992). The p53appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 does not affect the growth of cellswith endogenous p53. Thus, such constructs might be taken up by normalcells without adverse effects.

It is thus possible that the treatment of p53-associated cancers withwild type p53 may reduce the number of malignant cells. However, studiessuch as those described above are far from achieving such a goal, notleast because DNA transfection cannot be employed to introduce DNA intocancer cells within a patients' body.

C. Gene Therapy Techniques

There have been several experimental approaches to gene therapy proposedto date, but each suffer from their particular drawbacks (Mulligan,1993). As mentioned above, basic transfection methods exist in which DNAcontaining the gene of interest is introduced into cellsnon-biologically, for example, by permeabilizing the cell membranephysically or chemically. Naturally, this approach is limited to cellsthat can be temporarily removed from the body and can tolerate thecytotoxicity of the treatment, i.e. lymphocytes. Liposomes or proteinconjugates formed with certain lipids and amphophilic peptides can beused for transfection, but the efficiency of gene integration is stillvery low, on the order of one integration event per 1,000 to 100,000cells, and expression of transfected genes is often limited to days inproliferating cells or weeks in non proliferating cells. DNAtransfection is clearly, therefore, not a suitable method for cancertreatment.

A second approach capitalizes on the natural ability of viruses to entercells, bringing their own genetic material with them. Retroviruses havepromise as gene delivery vectors due to their ability to integrate theirgenes into the host genome, transferring a large amount of foreigngenetic material, infecting a broad spectrum of species and cell typesand of being packaged in special cell-lines. However, three majorproblems hamper the practical use of retrovirus vectors. First,retroviral infectivity depends on the availability of the viralreceptors on the target surface. Second, retroviruses only integrateefficiently into replicating cells. And finally, retroviruses aredifficult to concentrate and purify.

D. Adenovirus Constructs for Use in Gene Therapy

Human adenoviruses are double-stranded DNA tumor viruses with genomesizes of approximate 36 kb (Tooza, 1981). As a model system foreukaryotic gene expression, adenoviruses have been widely studied andwell characterized, which makes them an attractive system fordevelopment of adenovirus as a gene transfer system. This group ofviruses is easy to grow and manipulate, and they exhibit a broad hostrange in vitro and in vivo. In lytically infected cells, adenovirusesare capable of shutting off host protein synthesis, directing cellularmachineries to synthesize large quantities of viral proteins, andproducing copious amounts of virus.

The E1 region of the genome includes E1A and E1B which encode proteinsresponsible for transcription regulation of the viral genome, as well asa few cellular genes. E2 expression, including E2A and E2B, allowssynthesis of viral replicative functions, e.g. DNA-binding protein, DNApolymerase, and a terminal protein that primes replication. E3 geneproducts prevent cytolysis by cytotoxic T cells and tumor necrosisfactor and appear to be important for viral propagation. Functionsassociated with the E4 proteins include DNA replication, late geneexpression, and host cell shutoff. The late gene products include mostof the virion capsid proteins, and these are expressed only after mostof the processing of a single primary transcript from the major latepromoter has occurred. The major late promoter (MLP) exhibits highefficiency during the late phase of the infection (Stratford-Perricaudetand Perricaudet, 1991a).

As only a small portion of the viral genome appears to be required incis (Tooza, 1981), adenovirus-derived vectors offer excellent potentialfor the substitution of large DNA fragments when used in connection withcell lines such as 293 cells. Ad5-transformed human embryonic kidneycell line (Graham, et al., 1977) have been developed to provide theessential viral proteins in trans. The inventors thus reasoned that thecharacteristics of adenoviruses rendered them good candidates for use intargeting cancer cells in vivo (Grunhaus & Horwitz, 1992).

Particular advantages of an adenovirus system for delivering foreignproteins to a cell include (i) the ability to substitute relativelylarge pieces of viral DNA by foreign DNA; (ii) the structural stabilityof recombinant adenoviruses; (iii) the safety of adenoviraladministration to humans; and (iv) lack of any known association ofadenoviral infection with cancer or malignancies; (v) the ability toobtain high titers of the recombinant virus; and (vi) the highinfectivity of Adenovirus.

Further advantages of adenovirus vectors over retroviruses include thehigher levels of gene expression. Additionally, adenovirus replicationis independent of host gene replication, unlike retroviral sequences.Because adenovirus transforming genes in the E1 region can be readilydeleted and still provide efficient expression vectors, oncogenic riskfrom adenovirus vectors is thought to be negligible (Grunhaus & Horwitz,1992).

In general, adenovirus gene transfer systems are based upon recombinant,engineered adenovirus which is rendered replication-incompetent bydeletion of a portion of its genome, such as E1, and yet still retainsits competency for infection. Relatively large foreign proteins can beexpressed when additional deletions are made in the adenovirus genome.For example, adenoviruses deleted in both E1 and E3 regions are capableof carrying up to 10 Kb of foreign DNA and can be grown to high titersin 293 cells (Stratford-Perricaudet and Perricaudet, 1991a).Surprisingly persistent expression of transgenes following adenoviralinfection has also been reported.

Adenovirus-mediated gene transfer has recently been investigated as ameans of mediating gene transfer into eukaryotic cells and into wholeanimals. For example, in treating mice with the rare recessive geneticdisorder ornithine transcarbamylase (OTC) deficiency, it was found thatadenoviral constructs could be employed to supply the normal OTC enzyme.Unfortunately, the expression of normal levels of OTC was only achievedin 4 out of 17 instances (Stratford-Perricaudet et al., 1991b).Therefore, the defect was only partially corrected in most of the miceand led to no physiological or phenotypic change. These type of resultstherefore offer little encouragement for the use of adenoviral vectorsin cancer therapy.

Attempts to use adenovirus to transfer the gene for cystic fibrosistransmembrane conductance regulator (CFTR) into the pulmonary epitheliumof cotton rats have also been partially successful, although it has notbeen possible to assess the biological activity of the transferred genein the epithelium of the animals (Rosenfeld et al., 1992). Again, thesestudies demonstrated gene transfer and expression of the CFTR protein inlung airway cells but showed no physiologic effect. In the 1991 Sciencearticle, Rosenfeld et al. showed lung expression of α1-antitrypsinprotein but again showed no physiologic effect. In fact, they estimatedthat the levels of expression that they observed were only about 2% ofthe level required for protection of the lung in humans, i.e., far belowthat necessary for a physiologic effect.

The gene for human α₁-antitrypsin has been introduced into the liver ofnormal rats by intraportal injection, where it was expressed andresulted in the secretion of the introduced human protein into theplasma of these rats (Jaffe et al., 1992). However, and disappointingly,the levels that were obtained were not high enough to be of therapeuticvalue.

These type of results do not demonstrate that adenovirus is able todirect the expression of sufficient protein in recombinant cells toachieve a physiologically relevant effect, and they do not, therefore,suggest a usefulness of the adenovirus system for use in connection withcancer therapy. Furthermore, prior to the present invention, it wasthought that p53 could not be incorporated into a packaging cell, suchas those used to prepare adenovirus, as it would be toxic. As E1B ofadenovirus binds to p53, this was thought to be a further reason whyadenovirus and p53 technology could not be combined.

E. p53-Adenovirus Constructs and Tumor Suppression

The present invention provides cancer gene therapy with a new and moreeffective tumor suppressor vector. This recombinant virus exploits theadvantages of adenoviral vectors, such as high titer, broad targetrange, efficient transduction, and non-integration in target cells. Inone embodiment of the invention, a replication-defective,helper-independent adenovirus is created that expresses wild type p53(Ad5CMV-p53) under the control of the human cytomegalovirus promoter.

Control functions on expression vectors are often provided from viruseswhen expression is desired in mammalian cells. For example, commonlyused promoters are derived from polyoma, adenovirus 2 and simian virus40 (SV40). The early and late promoters of SV40 virus are particularlyuseful because both are obtained easily from the virus as a fragmentwhich also contains the SV40 viral origin of replication. Smaller orlarger SV40 fragments may also be used provided there is included theapproximately 250 bp sequence extending from the HindIII site toward theBglI site located in the viral origin of replication. Further, it isalso possible, and often desirable, to utilize promoter or controlsequences normally associated with the included gene sequence, providedsuch control sequences are compatible with the host cell systems.

An origin of replication may be provided by construction of the vectorto include an exogenous origin, such as may be derived from SV40 orother viral (e.g., polyoma, adeno, VSV, BPV) source, or may be providedby the host cell chromosomal replication mechanism. If the vector isintegrated into the host cell chromosome, the latter is oftensufficient.

The design and propagation of the preferred p53 adenovirus is diagramedin FIG. 1. In connection with this, an improved protocol has beendeveloped for propagating and identifying recombinant adenovirus(discussed below). After identification, the p53 recombinant adenoviruswas structurally confirmed by the PCR analysis, as indicated in FIG. 2.After isolation and confirmation of its structure, the p53 adenoviruswas used to infect human lung cancer cell line H358, which has ahomozygous p53 gene deletion. Western blots showed that the exogenousp53 protein was expressed at a high level (FIG. 4 and FIG. 5) and peakedat day 3 after infection (FIG. 6).

It was also shown in a p53 point mutation cell line H322 that the mutantp53 was down regulated by the expression of the exogenous p53. As anexperimental control, a virion (Ad5/RSV/GL2) that had a structuralsimilarity to that of Ad5CMV-p53 was used. This virion contained aluciferase CDNA driven by Rous sarcoma virus LTR promoter in theexpression cassette of the virion. Neither p53 expression nor change inactin expression was detected in cells infected by the virionAd5/RSV/GL2. Growth of the H358 cells infected with Ad5CMV-p53 wasgreatly inhibited in contrast to that of noninfected cells or the cellsinfected with the control virion (FIG. 7A). Growth of H322 cells wasalso greatly inhibited by the p53 virion (FIG. 7B), while that of humanlung cancer H460 cells containing wild-type p53 was less affected (FIG.7C).

Ad5CMV-p53 mediated a strong inhibitory-effect on lung cancer cellgrowth in vitro. Growth inhibition was not as evident when the cellswere infected with Ad5CMV-p53 at MOI lower than 1 PFU/cell, whereas, atMOI higher than 100 PFU/cell, cytotoxicity could be observed even withcontrol virus Ad5/RSV/GL2. In our studies, the optimal dose for growthrate studies was 10-50 PFU/cell. Within this dose range, cell growthinhibition was attributable to the expressed p53 protein.

Tests in nude mice demonstrated that tumorigenicity of theAd5CMV-p53-treated H358 cells was greatly inhibited. In a mouse model oforthotopic human lung cancer, the tumorigenic H226Br cells, with a pointmutation in p53, were inoculated intratracheally 3 days prior to thevirus treatment. Intratracheal instillation of Ad5CMV-p53 preventedtumor formation in this model system suggesting that the modifiedadenovirus is an efficient vector for mediating transfer and expressionof tumor suppressor genes in human cancer cells and that the Ad5CMV-p53virus may be further developed into a therapeutic agent for use incancer gene therapy.

Ad5CMV-p53 mediated a high level of expression of the p53 gene in humanlung cancer cells as demonstrated by Western blot analysis. Exogenousp53 protein was approximately 14 times more abundant than the endogenouswild-type p53 in H460 cells and about two to four times more abundantthan the β-actin internal control in H358 cells. The high level ofexpression may be attributed to (1) highly efficient gene transfer, (2)strong CMV promoter driving the p53 cDNA, and (3) adenoviral E1 enhancerenhancing the p53 CDNA transcription. The duration of p53 expressionafter infection was more than 15 days in H358 cells. However, there wasa rapid decrease in expression after postinfection day 5. PCR analysisof the DNA samples from the infected H358 cells showed a decrease of theviral DNA level with the decreased protein level, indicating the loss ofviral DNA during the continuous growth of cancer cells in vitro.

The decrease in p53 expression may also have resulted from cellularattenuation of the CMV promoter that controls p53 expression, since thephenomenon of host cell-mediated CMV promoter shut off has been reportedpreviously (Dai, et al., 1992). Adenoviral vectors are nonintegrativegene transfer vectors and therefore the duration of gene expressiondepends upon a number of factors, including the host cells, the genestransferred, and the relevant promoter. Crystal and co-workers showedlow level expression of the cystic fibrosis transmembrane conductanceregulator gene in cotton rat epithelial cells was detectable 6 weeksafter infection (Rosenfeld, et al., 1992). Perricaudet's laboratorydemonstrated minimal expression of minidystrophin gene in mdx mousemuscle lasted for more than 3 months after infection. The short-termhigh level expression of the wild-type p53 protein observed in thepresent study may have the beneficial effect of reducing possible sideeffects on normal cells following in vivo treatment with Ad5CMV-p53.

The studies disclosed herein indicate that the p53 recombinantadenovirus possesses properties of tumor suppression, which appear tooperate by restoring p53 protein function in tumor cells. These resultsprovide support for the use of the Ad5CMV-p53 virion as a therapeuticagent for cancer treatment.

F. DNA Damaging Agents

A wide variety of DNA damaging agents may be used with the presentinvention, such as, agents that directly crosslink DNA, agents thatintercalate into DNA, and agents that lead to chromosomal and mitoticaberrations by affecting nucleic acid synthesis.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged and are shown herein, to eventuate DNA damage leading to asynergistic antineoplastic combination. Agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis, and chromosomal segregation. Examples of thesecompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in clinicalsetting for the treatment of neoplasms these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m2at 21 day intervals for adriamycin, to 35-50 mg/m2 for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors, and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent-particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that these all of these factors effect a broad range ofdamage on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan in directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

G. p53 and Cisplatin Treatment

In an effort to determine the efficacy of a combination of genereplacement therapy and chemotherapy in human cancer, the inventorsexamined whether sequential administration of Ad-p53 and CDDP couldinduce apoptosis in vivo. Following 3 days of direct intratumoralinjection of Ad-p53 or intraperitoneal administration of CDDP, H358tumors implanted subcutaneously in nu/nu mice showed a modest slowing ofgrowth. However, if Ad-p53 and CDDP were simultaneously administered,tumors partially regressed and the tumor size remained statisticallysignificantly smaller than those in any of the other treatment groups.The growth inhibitory effect: was even more pronounced after twotreatment cycles (FIG. 13A). Histologic examination revealed a massivedestruction of tumor cells in the area where Ad-p53 was injected in micetreated with CDDP. In situ staining demonstrated many apoptotic cellsaround acellular spaces (FIG. 13B-E). In contrast, tumors treated withCDDP alone or Ad-p53 alone showed neither acellularity nor apoptoticareas.

The present invention describes a novel strategy for human gene therapycombined with conventional chemotherapy using a DNA crosslinking agent.Tumor cell resistance to chemotherapeutic drugs represents a majorproblem in clinical oncology. NSCLC accounts for at least 80% of thecases of lung cancer; patients with NSCLC are, however, generallyunresponsive to chemotherapy (Doyle, 1993). One goal of current cancerresearch is to find ways to improve the efficacy of gene replacementtherapy for cancer by investigating interaction between the gene productand chemotherapeutic drugs. The herpes simplex-thymidine kinase (HS-tK)gene, when delivered to brain tumors by a retroviral vector system,successfully induced susceptibility to the antiviral agent ganciclovir(Culver, et al., 1992). The HS-tK gene product is an exogenous viralenzyme, whereas the wt-p53 protein is expressed in normal tissues,suggesting that the modulation of chemoresistance by alterations inwt-p53 expression might be an alternative approach using a pathwaymediated by an endogenous genetic program.

An adenovirus system has potential advantages for gene delivery in vivo,such as ease of producing high titer virus, high infection efficiency,and infectivity for many types of cells. The stability and duration ofexpression of the introduced gene are still controversial, however. Forchemo-gene therapy, the levels of expression and the high infectivitymay be are more significant than the duration of expression, becausedrugs can kill infected cells within several days. The increase in p53levels in cells that are sensitive to chemotherapeutic drugs can occurwithin 6 hours after DNA-damaging stimuli (Fritsche, et al., 1993, Zhan,et al., 1993), although increased p53 DNA binding activity can bereversed over the course of 4 hours if the stimulus is removed (Tishler,et al., 1993). In the present model, the expression of the wt-p53 geneis driven independently by the cytomegalovirus promoter contained in anAd-p53 vector. Therefore, a high level of p53 expression can bemaintained even after cessation of drug exposure. The expression ofwt-p53 protein by Ad-p53 peaks at postinfection day 3 (14-fold greaterthan endogenous wild type) and decreases to a low level by day 9 (Zhang,et al., 1993). This suggests that a transiently high level of wt-p53expression is sufficient to initiate the cytotoxic program in the cancercell.

H. Patients and Treatment Protocols

The inventors propose that the regional delivery of adenoviral-p53 geneconstructs to lung cancer cells in patients with p53-linked cancers,such as unresectable obstructing endobronchial cancers, will be a veryefficient method for delivering a therapeutically effective gene tocounteract the clinical disease. The deliver of the p53 gene is to occurin combination with agents or factors that lead to DNA damage. Thiscombined approach is a significant improvement on current cancertherapies, for example the loss of sensitivity to cisplatin alone, whichrely on attempts to kill or remove the last cancer cell by effecting DNAdamage. As tumor cell dormancy is an established phenomenon, this makeseffective killing highly unlikely.

It is anticipated that the uptake of the adenovirus constructs by NSCLCcells will decrease the rate of proliferation of these cells, however,the present examples demonstrate that the combined use of a DNA damagingagent or factor with the p53 adenovirus leads to a profound diminutionof cell growth and tumor size, not shown with either factor alone. Thecompositions and methods disclosed herein, strongly portend an increasein the length of time the affected lung would remain expanded, preventregrowth of the tumor and division of tumor cells, and prolong thepatient's survival.

Patients with unresectable endobronchial tumor recurrence that ispartially or completely obstructing the airway and that have failed orare unable to receive external beam radiotherapy will be considered forthis combined protocol. Existing therapies for this condition offer onlyshort-term palliation. Most patients have recurred despite external beamradiotherapy. It may be possible to insert a brachytherapy catheter andadminister additional radiotherapy, intravenous administration of DNAdamaging agents. Patients receiving current treatments have a mediansurvival of 6 months. Patients failing brachytherapy would also beeligible to receive gene therapy. Tumor can be removed from the airwaywith the laser or biopsy forceps. This can be done in conjunction withinjection of the adenoviral constructs thus decreasing the volume thatmust be injected. The administration of the viral constructs would notpreclude the patient from receiving other palliative therapy if thetumor progresses.

I. Other Gene Transfer Techniques

Successful gene therapy generally requires the integration of a geneable to correct the genetic disorder into the host genome, where itwould co-exist and replicate with the host DNA and be expressed at alevel to compensate for the defective gene. Ideally, the disease wouldbe cured by one or a few treatments, with no serious side effects. Therehave been several approaches to gene therapy proposed to date, which maybe used with the present invention.

A first approach is to transfect DNA containing the gene of interestinto cells, e.g., by permeabilizing the cell membrane either chemicallyor physically. This approach is generally limited to cells that can betemporarily removed from the body and can tolerate the cytotoxicity ofthe treatment (i.e. lymphocytes). Liposomes or protein conjugates formedwith certain lipids and amphophilic peptides can be used for in vivotransfection (Stewart et al., 1992; Torchilin et al., 1992; Zhu et al.,1993), however present efficiency of gene integration is very low. It isestimated that the gene of interest integrates into the genome of onlyone cell in 1,000 to 100,000. In the absence of integration, expressionof the transfected gene is limited to several days in proliferatingcells or several weeks in non proliferating cells due to the degradationof the un-integrated DNAs.

A second approach capitalizes on the natural ability of viruses to entercells, bringing their own genetic material with them. Retroviruses havepromise as gene delivery vectors due to their ability to integrate theirgenes into the host genome, transferring a large amount of foreigngenetic material, infecting a broad spectrum of species and cell typesand of being packaged in special cell-lines (Miller, 1992).

A third method uses other viruses, such as adenovirus, herpes simplexvirues (HSV), cytomegalovirus (CMV), and adeno-associated virus (AAV),which are engineered to serve as vectors for gene transfer. Althoughsome viruses that can accept foreign genetic material are limited in thenumber of nucleotides they can accommodate and in the range of cellsthey infect, these viruses have been demonstrated to successfully effectgene expression. However, adenoviruses do not integrate their geneticmaterial into the host genome and therefore do not require hostreplication for gene expression, making them ideally suited for rapid,efficient, heterologous gene expression.

Even though the invention has been described with a certain degree ofparticularity, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art in light of theforegoing disclosure. Accordingly, it is intended that all suchalternatives, modifications, and variations which fall within the spiritand the scope of the invention be embraced by the defined claims.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Construction of p53 Expression Vector

This example describes the construction of a p53 expression vector. Thisvector is constructed as indicated and is used to replace the E1 region(1.3-9.2 m.u.) of the Adenovirus strain Ad5 genome and employed toconstruct the Adenovirus virion described in Example 2.

The p53 expression cassette shown in FIG. 1, which contains humancytomegalovirus (CMV) promoter (Boshart, et al., 1985), p53 cDNA, andSV40 early polyadenylation signal, was inserted between the Xba I andCla I sites of pXCJL1 (provided by Dr. Frank L. Graham, McMasterUniversity, Canada).

The genome size is about 35.4 kb, divided into 100 map units (1 m.u.0.35 kb). The p53 expression cassette replaced the E1 region (1.3-9.2m.u.) of the Ad5 genome.

Primer 1 has the sequence 5′-GGCCCACCCCCTTGGCTTC-3′ (SEQ ID NO:1) and islocated in the first intron downstream of the human CMV major IE genepromoter (Boshart, et al., 1985). Primer 2 has the sequence5′-TTGTAACCATTATAAGCTGC-3′ (SEQ ID NO:2) and is located in SV40 earlypolyadenylation signal. Both of the primers, 15-20 bp away from the p53cDNA insert at both ends, define a 1.40 kb PCR product. Primer 3 has thesequence 5′-TCGTTTCTCAGCAGCTGTTG-3′ (SEQ ID NO:3) and primer 4 has thesequence 5′-CATCTGAACTCAAAGCGTGG-3′ (SEQ ID NO:4) and are located at 11m.u. and 13.4 m.u. of the Ad5 genome, respectively, which define a 0.86kb viral-genome specific PCR product.

EXAMPLE 2 Generation and Propagation of Recombinant p53 Adenovirus

This example describes one method suitable for generatinghelper-independent recombinant adenoviruses expressing p53. Themolecular strategy employed to produce recombinant adenovirus is basedupon the fact that, due to the packaging limit of adenovirus, pJM17cannot form virus on its own. Therefore, homologous recombinationbetween the p53 expression vector plasmid and pJM17 within a transfectedcell results in a viable virus that can be packaged only in cells whichexpress the necessary adenoviral proteins.

The method of this example utilizes 293 cells as host cells to propagateviruses that contain substitutions of heterologous DNA expressioncassettes at the E1 or E3 regions. This process requires cotransfectionof DNA into 293 cells. The transfection largely determines efficiency ofviral propagation. The method used for transfection of DNA into 293cells prior to the present invention was usually calcium-phosphate/DNAcoprecipitation (Graham and van der Eb, 1973). However, this methodtogether with the plaque assay is relatively difficult and typicallyresults in low efficiency of viral propagation. As illustrated in thisexample, transfection and subsequent identification of infected cellswere significantly improved by using liposome-mediated transfection,when identifying the transfected cells by cytopathic effect (CPE).

The 293 cell line was maintained in Dulbecco's modified minimalessential medium supplemented with 10% heat-inactivated horse serum. Thep53 expression vector and the plasmid pJM17 (McGrory, et al., 1988) forhomologous recombination were cotransfected into 293 cells byDOTAP-mediated transfection according to the manufacture's protocol(Boehringer Mannheim Biochemicals, 1992). This is schematically shown inFIG. 1.

The 293 cells (passage 35, 60% confluency) were inoculated 24 hoursprior to the transfection in either 60 mm dishes or 24-well plates. Thecells in each well were transfected with: 30 μl. DOTAP, 2 μg of p53expression vector, and 3 μg of plasmid pJM17. After transfection cellswere fed with the MEM medium every 2-3 days until the onset of CPE.

EXAMPLE 3 Confirming the Identity of Recombinant Adenovirus

This example illustrates a new polymerase chain reaction (PCR) assay forconfirming the identity of recombinant virions following cotransfectionof the appropriate cell line.

Aliquots of cell culture supernatants (50 to 370 μl) were collected fromthe test plates, treated with proteinase K (50 μg/ml with 0.5% SDS and20 mM EDTA) at 56° C. for 1 hour, extracted with phenol-chloroform, andthe nucleic acids were ethanol precipitated. The DNA pellets wereresuspended in 20 μl dH₂O and used as template for PCR amplification.The relative locations of the PCR primers and their sequences aredepicted in FIG. 1 and are SEQ ID NOS:1, 2, 3 and 4, respectively. ThecDNA insert-specific primers define a 1.4 kb PCR product and the viralgenome-specific primers define a 0.86 kb PCR product. The PCR reactionswere carried out in a 50 μl volume containing 4 mM MgCl₂, 50 mM KCl,0.1% triton X-100, 200 μM each of dTPs, 10 mM Tris-Cl (pH 9.0), 2 μM ofeach primer, and 1.0 unit of Taq polymerase (Promega). The reactionswere carried out at 94° C., 0.5 min, 56° C., 0.5 min, and 72° C., 1 minfor 30 cycles.

In order to simplify the procedure of identification of newly propagatedrecombinant virus, a direct PCR assay on DNA samples from cell culturesupernatant was developed. Aliquots (50 or 370 μl) of the cell mediumsupernatant with CPE were treated with proteinase K andphenol/chloroform extraction. After ethanol precipitation, the DNAsamples were analyzed using PCR employing two pairs of primers toamplify insert-specific and viral-genome-specific sequences. The PCRprimer targets and their sequences are depicted in FIG. 1. Primers 1, 2,3 and 4 are represented by SEQ ID NOS:1, 2, 3 and 4, respectively.

As a result, a 1.4 kb cDNA insert and a 0.86 kb viral genome fragmentwere amplified from the expression vector (positive control) and the DNAsamples of the positive cell culture (FIG. 2B, lane 1 and 4,respectively). Only the 0.86 kb fragment was amplified from the DNAsample of Ad5/RSV/GL2 virus (negative control, lane 2). No amplifiedbands appeared from PCR reactions that used either untreated positivecell culture medium supernatant (lane 3).

These results indicated that adenoviruses released into cell culturemedium are detectable by PCR, using as little as 50 μL of the cellculture medium supernatant for preparing DNA templates. These resultswill allow development of a quantitative method for using this techniqueto determine adenovirus titers, traditionally done by plaque assays.

The wild-type sequence of the p53 cDNA in the Ad5CMV-p53 virus wasconfirmed by dideoxy DNA sequencing on the CsCl-gradient-purified viralDNA. The control virus Ad5/RSV/GL2, generated in a similar manner, has astructure similar to that of Ad5CMV-p53 except a Rous sarcoma viralpromoter and luciferase cDNA were used in its expression cassette. Therecombinant adenovirus that carries a E. coli β-galactosidase gene(LacZ), Ad5CMV-LacZ, also has a structure similar to that of Ad5CMV-p53,and is obtainable as disclosed in Zhang et al. and from Dr. Frank L.Graham (please see Graham, et al., 1991).

Viral stock, titer, and infection. Individual clones of the Ad5CMV-p53,Ad5/RSV/GL2, and Ad5CMV-LacZ viruses were obtained byplaque-purification according to the method of Graham and Prevec (1991).Single viral clones were propagated in 293 cells. The culture medium ofthe 293 cells showing the completed cytopathic effect was collected andcentrifuged at 1000×g for 10 min. The pooled supernatants were aliquotedand stored at −20° C. as viral stocks. The viral titers were determinedby plaque assays (Graham and Prevec, 1991). Infections of the cell lineswere carried out by addition of the viral solutions (0.5 ml per 60-mmdish) to cell monolayers and incubation at room temperature for 30 minwith brief agitation every 5 min. This was followed by the addition ofculture medium and the return of the infected cells to the 37° C.incubator.

The gene transfer efficiency of the recombinant adenoviruses was alsoevaluated using Ad5CMV-LacZ in a variety of cell lines such as H226Br,H322, H460, HeLa, Hep G2, LM2, and Vero. By X-gal staining, all of thecell lines were stained 97-100′ blue after infection with Ad5CMV-LacZ atan MOI of 30 PFU/cell.

EXAMPLE 4 Ad5CMV-p53-Directed p53 Gene Expression in Human Lung CancerCells

This example describes the use of recombinant p53 adenovirus to infecthuman lung cancer cells with a homozygous p53 gene deletion. The resultsshow that growth of these cells and expression of mutant p53 wassuppressed, indicating the potential of the Ad5CMV-p53 virion as auseful agent for control of metastatic cells.

Immunohistochemistry was performed on infected cell monolayers that werefixed with 3.8% formalin and treated with 3% H₂O₂ in methanol for 5 min.Immunohistochemical analysis was performed using Vectastain Elite kit(Vector, Burlingame, Calif.). The primary antibody used was anti-p53antibody PAb 1801 (Oncogene Science, Manhasset, N.Y.); MOPC-21 (OrganonTeknika Corp., West Chester, Pa.) was used as a negative control. Thesecond antibody was an avidin-labeled anti-mouse IgG (Vector). Thebiotinylated horseradish peroxidase ABC complex reagent was used todetect the antigen-antibody complex. Finally the cells werecounterstained with Harris hematoxylin (Sigma) and mounted with Cytoseal60 (Stephens Scientific, Riverdale, N.J.).

Immunohistochemical analysis of the infected cell lines was performed toexamine the in situ expression of p53 expression driven by the CMVpromoter of the Ad5CMV-53 virus. In the H358 cell line, which has ahomozygous deletion of p53, the p53 gene was transferred with 97-100%efficiency, as detected by immunohistochemical analysis, when the cellswere infected with Ad5CMV-p53 at a multiplicity of infection of 30-50plaque-forming units (PFU)/cell (FIG. 4).

The high transfer efficiency of recombinant adenovirus was confirmed byAd5CMV-LacZ, a virus which carries the LacZ gene transcribed by thehuman CMV IE promoter. At an MOI of 30-50 PFU/cell, all of the cellsexamined, including HeLa, Hep G2, LM2, and the human NSCLC cancer delllines were 97-100% positive for b-galactosidase activity by X-galstaining. These results indicate that adenoviral vectors are anefficient vehicle for gene transfer into human cancer cells.

Western blotting analysis was performed on total cell lysates preparedby lysing monolayer cells in dishes with SDS-PAGE sample buffer (0.5 mlper 60-mm dish) after rinsing the cells with phosphate-buffered saline(PBS). For SDS-PAGE analysis lanes were loaded with cell lysatesequivalent to 5×10⁴ cells (10-15 ml). The proteins in the gel weretransferred to Hybond™-ECL membrane (Amersham, Arlington Heights, Ill.).The membranes were blocked with 0.5% dry milk in PBS and probed with theprimary antibodies: mouse anti-human p53 monoclonal antibody PAb 1801and mouse anti-human β-actin monoclonal antibody (Amersham), washed andprobed with the secondary antibody: horseradish peroxidase-conjugatedrabbit anti-mouse IgG (Pierce Chemical Co., Rockford, Ill.). Themembranes were developed according to the Amersham's enhancedchemiluminescence protocol. Relative quantities of the exogenous p53expressed were determined by densitometer (Molecular Dynamics Inc.,Sunnyvale, Calif.).

Western blots showed the exogenous p53 protein was expressed at a highlevel (FIG. 5A lanes 2, 3 and 5, 6). The protein peaked at day 3 afterinfection (FIG. 6, insert, 0.5 days to 3 days). As a control, a virionwith a structure similar to the recombinant Ad5CMV-p53 of Example 1 wasconstructed. This virion contains a luciferase cDNA driven by RousSarcoma Virus LTR promoter in the expression cassette of the virion.Neither p53 expression nor change in actin expression was detected inthe cells infected by the virion Ad5/RSV/GL2.

The recombinant p53 adenovirus was used to infect three human lungsNSCLC cell lines: cell line H358, which has a homozygous deletion of thep53 gene, cell line H322, which has a point mutation of the p53 gene atcodon 248 (G to T), and cell line H460, which has a wild-type p53 gene.The growth rate of human NSCLC cells was determined following theinoculation of H322 and H460 (1×10⁵) or H358 (2×10⁵) in 60-mm culturedishes 24 h before viral infection. The cells were infected with theviruses at a multiplicity of infection (MOI) of 10 PFU/cell. Culturemedium was used for the mock infection control. Triplet cultures of eachcell line with different treatments were counted daily for days 1-6after infection.

Growth of the H358 cells infected with Ad5CMV-p53 was greatly inhibitedin contrast to that of noninfected cells or the cells infected with thecontrol virion (FIG. 7A). Growth of H322 cells was also greatlyinhibited by the p53 virion (FIG. 7B), while that of human lung cancerH460 cells containing wild type p53 was affected to a lesser degree(FIG. 7C). Growth of the Ad5CMV-p53 virus-infected H358 cells wasinhibited 79%, whereas that of noninfected cells or the cells infectedwith the control virus were not inhibited. Growth of cell line H322,which has a point mutation in p53, was inhibited 72% by Ad5CMV-p53,while that of cell line H460 containing wild-type p53 was less affected(28% inhibition).

The results indicate that the p53 recombinant adenovirus possessesproperties of tumor suppression, working through restoration of the p53protein function in tumor cells.

EXAMPLE 5 Ad5CMV-p53 in the Treatment of p53 Deficient Cells

The present example concerns the use of recombinant p53 adenovirus torestore growth suppression of tumor cells in vitro and thus to treat themalignant or metastatic growth of cells. It describes some of the waysin which the present invention is envisioned to be of use in thetreatment of cancer via adenovirus-mediated gene therapy.

H358 cells were infected with Ad5CMV-p53 and Ad5/RSV/GL2 at a MOI of 10PFU/cell. An equal amount of cells were treated with medium as a mockinfection. Twenty-four hours after infection, the treated cells wereharvested and rinsed twice with PBS. For each treatment, three million(3×10⁶) cells in a volume of 0.1 ml were injected s.c. to each nudemouse (Harlan Co., Houston, Tex.). Five mice were used for eachtreatment. Mice were irradiated (300 cGy, ⁶⁰Co) before injection andexamined weekly after injection. Tumor formation was evaluated at theend of a 6-week period and tumor volume was calculated by assuming aspherical shape with the average tumor diameter calculated as the squareroot of the product of cross-sectional diameters.

To determine the inhibitory effect on tumorigenicity mediated byAd5CMV-p53 nude mice were injected s.c. with H358 cells (a humanNSCLC-type cell) to induce neoplastic growth. Each mouse received oneinjection of cells that had been infected with Ad5CMV-p53 or Ad5/RSV/GL2at 10 PFU/cell for 24 h. H358 cells treated with medium alone were usedas mock-infected controls. Tumors, first palpable at postinjection day14, were induced only by the mock- or control virus-infected cells asdemonstrated in Table I: TABLE I Effect of Ad5CMV-p53 on tumorigenicityof H358 in nude mice^(a) No. of Tumors/ Mean Volume Treatment No. ofMice (%) (mm³ ± SD) Medium 4/5 (80) 37 ± 12 Ad5/RSV/GL2 3/4 (75) 30 ± 14Ad5CMV-p53 0/4 (0)  —^(a)The treated H358 cells were injected s.c. at 2 × 10⁶ cells/mouse.Tumor sizes were determined at the end of a 6-week period.

As shown in Table 1 mice that received Ad5CMV-p53-treated cells did notdevelop tumors. The tumors at the end of a 6-week period were 4-10 mm indiameter. This study was initiated with five mice per group; one mouseeach in the Ad5CMV-p53 or Ad5/RSV/GL2 group failed to complete thestudy. The early deaths were presumably due to nosocomial infection.

EXAMPLE 6 Ad5CMV-p53 in the Treatment of Lung Cancer

The present example concerns the use of recombinant p53 adenovirus torestore growth suppression of tumor cells in vivo and thus to treatcancers in animals. It describes some of the ways in which the presentinvention is envisioned to be of use in the treatment of cancer viaadenovirus-mediated gene therapy.

The efficacy of Ad5CMV-p53 in inhibiting tumorigenicity was furtherevaluated in the mouse model of orthotopic human lung cancer. Since H358and H322 cells did not produce tumors in this model, cell line H226Brwas used. This cell line has a squamous lung cancer origin andmetastasized from lung to brain. H226br has a point mutation (ATC toGTC) at exon 7, codon 254, of the p53 gene and is tumorigenic in mice.

The procedure for tests in the mouse model of orthotopic human lungcancer has been previously described (Georges, et al., 1993). Briefly,nude mice treated with radiation (300 cGy, ⁶⁰Co) were inoculated withH226Br cells by intratracheal instillation. Each mouse received 2×10⁶cells in a volume of 0.1 ml PBS. Three days after inoculation, 10 miceper group were treated with 0.1 ml of viruses or vehicle (PBS) byintratracheal instillation once a day for two days. The virus dosageused was 5×10⁷ Ad5CMV-p53 or Ad5/RSV/GL2 per mouse. The mice wereeuthanized at the end of a 6-week period. Tumor formation was evaluatedby dissecting the lung and mediastinum tissues and measuring the tumorsize. The tumors were confirmed by histologic analysis of the sectionsof the tumor mass.

The irradiated nude mice were inoculated with 2×10⁶H226Br cells/mouse byintratracheal instillation. Three days after inoculation, each of themice (8-10 mice per group) were treated with 0.1 ml of either Ad5CMV-p53or Ad5/RSV/GL2 or vehicle (PBS) by intratracheal instillation once a dayfor two days. The virus dosage used was 5×10⁷ PFU/mouse. Tumor formationwas evaluated at the end of a 6-week period by dissecting the lung andmediastinum tissues and measuring the tumor size. A flow chart of theprocedure is depicted in FIG. 7, with representative samples ofdissection demonstrated in FIG. 8. The detected tumors were confirmed byhistologic analysis. The data of tumor measurements are summarized inTable II: TABLE II Effect of Ad5CMV-p53 on tumorigenicity of H226Br inmouse model of orthotopic human lung cancer^(a) No. mice with Tumors/Mean Volume Treatment Total Mice (%) (mm³ ± SD) Vehicle 7/10 (70) 30 ±8.4 Ad5/RSV/GL2 8/10 (80) 25 ± 6.9 Ad5CMV-p53  2/8 (25)  8 ± 3.3^(b)^(a)Mice were inoculated with 2 × 10⁶ H226Br cells/mouseintratracheally. On the 3rd day postinoculation, the mice were giveneither vehicle or viruses (5 × 10⁷ each in 0.1 ml) intratracheally oncea day for 2 days. Tumor formation was evaluated at the end of a 6-weekperiod.^(b)p < 0.05 by two-way analysis of variance when compared to the groupsreceiving vehicle (PBS) or virus control.

Only 25% of the Ad5CMV-p53-treated mice formed tumors, whereas in thevehicle or Ad5/RSV/GL2 control group, 70-80% of the treated mice formedtumors. The average tumor size of the Ad5CMV-p53 group was significantlysmaller than those of the control groups. These results indicate thatAd5CMV-p53 can prevent H226Br from forming tumors in the mouse model oforthotopic human lung cancer.

EXAMPLE 7 Synergism Between p53 and DNA Damage

The biochemical features of programmed cell death (apoptosis) show acharacteristic pattern of DNA fragmentation resulting from cleavage ofnuclear DNA. Recent studies have demonstrated that induction ofapoptosis by chemotherapeutic drugs or ionizing radiation may be relatedto the status of the p53 gene and that DNA-damaging stimuli are able toelevate intracellular p53 protein levels in cells that are in theprocess of apoptosis (Lowe, et al., 1993, Clarke, et al., 1993,Fritsche, et al., 1993, Harper, et al., 1993, E1-Deiry, et al., 1993).Inhibition of the cell cycle at the G₁ phase by increased levels of thewild-type p53 (wt-p53) protein allows more time for DNA repair; ifoptimal repair is impossible, p53 may trigger programmed cell death.Thus, p53 may contribute to the induction of apoptotic tumor cell deathby chemotherapeutic agents.

Inactivation of the p53 gene by missense mutation or deletion is themost common genetic alteration in human cancers (Levine, et al., 1991,Hollstein, et al., 1991). The loss of p53 function has been reported toenhance cellular resistance to a variety of chemotherapeutic agents(Lowe, et al., 1993). The inventors studies showed that human non-smallcell lung cancer (NSCLC) H358 cells, in which both alleles of p53 aredeleted, were resistant to chemotherapeutic drugs, whereas cell lineWTH226b, which has endogenous wt-p53, readily showed apoptotic celldeath 1.6 hours after treatment with cisplatin (CDDP) and etoposide(VP-16) (T. Fujiwara, E. A. Grimm, T. Mukhopadhyay, J. A. Roth,unpublished data). Therefore, the inventors sought to determine whetherthe introduction of the wt-p53 gene into H358 cells by an adenoviralvector could increase the cell's sensitivity to the DNA crosslinkingagent CDDP in vitro and in vivo.

Materials and Methods

H358 cells were kindly provided by A. Gazdar and J. Minna (Takahashi, etal., 1989).

Adenovirus Vectors

The construction and identification of a recombinant adenovirus vectorthat contains the cDNA that encodes human wt-p53 (Ad-p53) or luciferase(Ad-Luc) were previously reported (Zhang, et al., 1993). Briefly, thep53 expression cassette that contains human cytomegalovirus promoter,wt-p53 cDNA, and SV40 early polyadenylation signal, was inserted betweenthe XbaI and ClaI sites of pXCJL.1. The p53 shuttle vector and therecombinant plasmid pJM17 were cotransfected into 293 cells(Ad5-transformed human embryonic kidney cell line) by aliposome-mediated technique. The culture supernatant of 293 cellsshowing the complete cytopathic effect was collected and used forsubsequent infections. The control Ad-Luc virus was generated in asimilar manner. Ad-p53 and Ad-Luc viruses were propagated in 293 cells.The presence of replication competent virus was excluded by HeLa cellassays. The viral titers were determined by plaque assays (Graham, etal., 1991).

Detection of Nucleosomal DNA Fragmentation

DNA was isolated from parental, Ad-Luc-infected, and Ad-p53-infectedcells that did or did not receive CDDP treatment, by incubating cells at55° C. for 6 hours in lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA,100 mM NaCl, 1% SDS, and 50 μg/ml proteinase K). DNA was extracted twicewith equal volumes of phenol and once with chloroform-isoamylalcohol(24:1) and then precipitated in ethanol. Samples were subjected toelectrophoresis on a 1.5% agarose gel, and visualized by ethidiumbromide staining.

TdT-mediated dUTP nick end labeling was performed according to aprocedure previously reported (Gavrieli, et al., 1992). Monolayer cellswere treated with 0.01% NP-40. The slides were immersed in TdT buffer(30 mM Tris-HCl, pH 7.2; 140 mM sodium cacodylate; 1 mM cobalt chloride)and incubated with biotinylated dUTP (Boehringer Mannheim, Indianapolis,Ind.) and TdT at 37° C. for 45 min. The slides were covered with 2%bovine serum albumin for 10 min and incubated with avidin-biotin complex(Vectastain Elite Kit; Vector Laboratories, Burlingame, Calif.) for 30min. The calorimetric detection was performed by usingdiamino-benzidine.

Results

H358 cells were transduced in vitro with the human wt-p53 cDNA byexposure to Ad-p53. Western blot analysis showed a high level of wt-p53protein expression as early as 24 hours after infection with Ad-p53, butno wt-p53 was detected in parental (uninfected) cells or control cellsinfected with Ad-Luc (data not shown). Concurrent immunohistochemicalevaluation demonstrated detectable wt-p53 protein in more than 80% ofinfected cells, suggesting that the transfer and expression of p53 byAD-p53 was highly efficient (data not shown).

Continuous exposure of Ad-p53-infected H358 cells to CDDP reduced theirviability rapidly, whereas significant cell death for parental andAd-Luc-infected cells occurred only after 72 hours of exposure to CDDP(FIG. 10A). Loss of viability was greatly enhanced in cells transducedwith Ad-p53. Moreover, the reduction of viability could be observed evenwhen cells were maintained in drug-free medium after 24 hours ofexposure, suggesting that lethal damage could be induced within 24 hours(FIG. 10B). The sensitivity of wt-p53-transduced H358 cells to CDDP wasdose dependent (FIG. 10C).

An internucleosomal DNA ladder indicative of DNA fragmentation wasevident in cells expressing wt-p53 after 24 hours of exposure to CDDP;parental and Ad-Luc-infected cells, however, did not show DNAfragmentation (FIG. 11A). Terminal deoxynucleotidyl transferase(TdT)-mediated 2′-deoxyuridine-5′-triphosphate (dUTP)-biotin nick endlabeling, which detects DNA fragmentation characteristic of apoptosis insitu, showed many apoptotic cells in Ad-p53-infected cells treated withCDDP for 24 hours as shown in FIG. 11 G which demonstrates darklystaining nuclei and nuclear fragments not present in FIGS. 11B-F.

Introduction of wt-p53 is known to induce apoptosis in some types oftumor cell lines with deleted or mutated p53 (Yonish-Rouach, et al.,1991, Shaw, et al., 1992, Ramqvist, et al., 1993). However,overexpression of wt-p53 alone could not promote DNA fragmentation inthe p53-negative H358 cell line (FIG. 11), although their growth wassuppressed by Ad-p53 (FIG. 10). This is compatible with the inventorsprevious observations showing that stable H358 clones could be obtainedafter retrovirus-mediated wt-p53 transfer and that the clones grew moreslowly than parental cells (Cai, et al., 1993).

The potential therapeutic efficacy of the combination of Ad-p53 and CDDPwas evaluated in terms of the relative change in volume of H358spheroids. The multicellular tumor spheroid model exhibits in vitro ahistologic structure similar to that of primary tumors andmicrometastases. Treatment with CDDP caused a reduction of relativevolume in Ad-p53-infected H358 spheroids, but had no significant effecton parental or Ad-Luc-infected spheroids (FIG. 12A). In situTdT-mediated dUTP labeling showed many cells in the process of apoptosison the surface of Ad-p53-infected spheroids, while no apoptotic cellswere seen on spheroids not infected with Ad-p53 (FIG. 12B-E). Theinventors have previously reported that retroviral-mediated wt-p53expression inhibited growth of H322a spheroids induced by transforminggrowth factor α (TGF-α) (Fujiwara, et al., 1993). The retroviral vectorcould not infect H358 spheroids, however, because cells in thesespheroids did not proliferate rapidly in response to exogenous TGF-α.The finding that exposure to CDDP reduced the size of H358 spheroidsinfected with Ad-p53 by inducing apoptosis on the surface suggests thatAd-p53 infects nonproliferating cells and that CDDP initiates theapoptotic process in quiescent cells.

EXAMPLE 8 Using p53 and DNA Damaging Agents in Treatment Regimens

An animal models has been employed as part of pre-clinical trials, asdescribed hereinbelow and in Examples 5, 6 and 7. Patients for whom themedical indication for adenovirus-mediated gene transfer treatment hasbeen established may be tested for the presence of antibodies directedagainst adenovirus. If antibodies are present and the patient has ahistory of allergy to either pharmacological or naturally occurringsubstances, application of a test dose of on the order of 103 to 106recombinant adenovirus under close clinical observation would beindicated.

For the treatment of cancer using Ad5CMV-p53, recombinant adenovirusexpressing p53 under the control of suitable promoter/enhancer elements,such as the CMV promoter, would be prepared and purified according to amethod that would be acceptable to the Food and Drug Administration(FDA) for administration to human subjects. Such methods include, butare not limited to, cesium chloride density gradient centrifugation,followed by testing for efficacy and purity.

Two basic methods are considered to be suitable for p53 adenovirustreatment methods, a direct or local administration and a more generaladministration. The present methods are suitable for treating any of thevariety of different cancers known to be connected with p53 mutations.In regard to general administration, a simple intravenous injection ofadenovirus has been shown to be sufficient to result in viral infectionof tissues at sites distant from the injection (Stratford-Perricaudet etal., 1991b), and is thus suitable for the treatment of all p53-linkedmalignancies. The virus may be administered to patients by means ofintravenous administration in any pharmacologically acceptable solution,or as an infusion over a period of time. Generally speaking, it isbelieved that the effective number of functional virus particles to beadministered would range from 1×10¹⁰ to 5×10¹².

Also, particularly where lung cancer is concerned, more direct physicaltargeting of the recombinant adenovirus could be employed if desired, inan analogous manner to the intratracheal administration of the cysticfibrosis transmembrane conductance regulator (Rosenfeld et al., 1992).This would result in the delivery of recombinant p53 adenovirus closerto the site of the target cells.

Methods

In situ dUTP labeling with TdT for detection of apoptosis. H358spheroids were fixed on day 3 and stained as described in Example 7.Briefly, labeled TdT probes were contacted to slides immersed in TdTbuffer and incubated with biotinylated dUTP and TdT at 37° C. for 45min. The slides were covered with 2% bovine serum albumin for 10 min andincubated with avidin-biotin complex for 30 min. The calorimetricdetection was performed using diamino-benzidine.

Induction of apoptosis by CDDP after in vivo infection with Ad-p53. H358cells (5×10⁶) in 0.1 ml Hank's balanced salt solution were injectedsubcutaneously into the right flank of BALB/c female nu/nu mice. Thirtydays later, 200 μl of medium alone or medium containing Ad-Luc (10⁸PFU/ml) or Ad-p53 (10′ PFU/ml) was injected into tumors with a diameterof 5 to 6 mm. Intratumoral injection (100 μl) and peritumoral injectionin two opposite sites (50 μl each) were performed. CDDP (3 mg/kg) orcontrol physiological saline was given intraperitoneally. (A) Tumorvolume changes. The tumors were measured with calipers in twoperpendicular diameters without the knowledge of the treatment groups,and a tumor volume was calculated by assuming a spherical shape with theaverage tumor diameter calculated as the square root of the product ofcross-sectional diameters. Five mice were used for each treatment groupand the mean+/−SE is shown. The data was analyzed using the Student'st-test. The arrow shows the day of treatment. Two independentdeterminations are shown. p<0.05 from day 5 in test 1; p<0.05 from day 7in test 2. Histologic study using the TdT-mediated biotin-dUTP labelingtechnique. Tumors were harvested 5 days after the beginning of treatmentand immediately embedded into O. C. T. compound. Frozen tissues were cutin a cryostat at 5-μm thicknesses. The sections were treated with 1μg/ml proteinase K and stained as described above. All animal care wasin accordance with the UT M.D. Anderson Institutional Animal Care andUse Committee.

Results

To demonstrate the in vivo efficacy of the methods and compositionsefficacy of a combination of gene replacement therapy and chemotherapyin human cancer, the inventors examined whether sequentialadministration of Ad-p53 and CDDP could induce apoptosis in vivo.Following 3 days of direct intratumoral injection of Ad-p53 orintraperitoneal administration of CDDP, H358 tumors implantedsubcutaneously in nu/nu mice showed a modest slowing of growth. However,if Ad-p53 and CDDP were simultaneously administered, tumors partiallyregressed and the tumor size remained statistically significantlysmaller than those in any of the other treatment groups. The growthinhibitory effect was even more pronounced after two treatment cycles(FIG. 13A). Histologic examination revealed a massive destruction oftumor cells in the area where Ad-p53 was injected in mice treated withCDDP. In situ staining demonstrated many apoptotic cells aroundacellular spaces (FIG. 13B-E). In contrast, tumors treated with CDDPalone or Ad-p53 alone showed neither acellularity nor apoptotic areas.

In more detail, preferred treatment protocols may be developed along thefollowing lines. Patients may first undergo bronchoscopy to assess thedegree of obstruction. As much gross tumor as possible should beresected endoscopically. Patients should preferably undergo bronchoscopyunder topical or general anesthesia. A Stifcor™ transbronchialaspiration needle (21 g) will be passed through the biopsy channel ofthe bronchoscope. The residual tumor site would then be injected withthe p53 adenovirus in a small volume such as about 10 ml or less.

In any event, since the adenovirus employed will be replicationincompetent, no deleterious effect of the virus itself on subject healthis anticipated. However, patients would remain hospitalized during thetreatment for at least 48 hours to monitor acute and delayed adversereactions. Safety-related concerns of the use of replication deficientadenovirus as a gene transfer vehicle in humans have been addressed inthe past (Rosenfeld et al., 1992; Jaffe et al., 1992), but the dose ofadenovirus to be administered should be appropriately monitored so as tofurther minimize the chance of untoward side effects.

There are various criteria that one should consider as presenting theexistence of a need for response or the existence of toxicity. To assistin determining the existence of toxicity, the tumor bed should bephotographed prior to a course of therapy. The longest diameter and itsperpendicular will be measured. Size will be reported as the product ofthe diameters. From these data, one can calculate from these numbers therate of regrowth of the tumor.

The time to progression can also be measured from the first observationwith reduction in tumor bulk until there is evidence of progressivedisease. Progressive Disease is defined as an increase of ≧25% in thesum of the products of the diameters of the measured lesion. Patientsmust have received at least two courses of therapy before a designationof progression is made. The survival of patients will be measured fromentry into protocol.

Follow-up examinations would include all those routinely employed incancer therapy, including monitoring clinical signs and taking biopsiesfor standard and molecular biological analysis in which the pattern ofexpression of various p53 genes could be assessed. This would alsosupply information about the number of cells that have taken up thetransferred gene and about the relative promoter strength in vivo. Basedon the data obtained adjustments to the treatment may be desirable.These adjustments might include adenovirus constructs that use differentpromoters or a change in the number of pfu injected to ensure ainfection of more, or all, tumor cells without unphysiologicaloverexpression of the recombinant genes.

It is contemplated that the expression of exogenous genes transferred invivo by adenovirus can persist for extended periods of time.Therapeutically effective long-term expression of virally transferredexogenous genes will have to be addressed on a case by case basis.Marker genes are limited in their usefulness to assess therapeuticallyrelevant persistence of gene expression as the expression levelsrequired for the amelioration of any given genetic disorder might differconsiderably from the level required to completely cure another disease.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims. All claimedmatter and methods can be made and executed without undueexperimentation.

REFERENCES

The following references to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method of killing a cell, comprising contacting a cell with a p53protein or gene and a DNA damaging agent in a combined amount effectiveto kill said cell.
 2. The method of claim 1, wherein said cell iscontacted with a p53 protein or gene in combination with X-rayradiation, UV-irradiation, γ-irradiation, microwaves, adriamycin,5-fluorouracil, etoposide, camptothecin, actimomycin-D, mitomycin C, orcisplatin.
 3. The method of claim 2, wherein said cell is contacted witha p53 protein or gene in combination with cisplatin.
 4. The method ofclaim 1, wherein said cell is contacted with a recombinant vector thatexpresses a p53 protein in said cell in combination with a DNA damagingagent.
 5. The method of claim 4, wherein said p53-expressing recombinantvector is a naked DNA plasmid, a plasmid within a liposome, a retroviralvector, an AAV vector, or a recombinant adenoviral vector.
 6. The methodof claim 5, wherein said p53-expressing recombinant vector is arecombinant adenoviral vector.
 7. The method of claim 6, wherein saidp53-expressing recombinant vector is a recombinant adenoviral vectorcomprising a p53 expression region positioned under the control of thecytomegalovirus IE promoter.
 8. The method of claim 6, wherein saidrecombinant adenoviral vector comprises a p53 expression region, thecytomegalovirus IE promoter and the SV40 early polyadenylation signal.9. The method of claim 6, wherein at least one gene essential foradenovirus replication is deleted from said adenovirus vector constructand the p53 expression region is introduced in its place.
 10. The methodof claim 9, wherein the E1A and E1B regions of the adenovirus vector aredeleted and the p53 expression region is introduced in their place. 11.The method of claim 6, wherein said recombinant adenoviral vector ispresent within a recombinant adenovirus.
 12. The method of claim 1,wherein said cell is first contacted with a p53 protein or gene and issubsequently contacted with a DNA damaging agent.
 13. The method ofclaim 1, wherein said cell is first contacted with a DNA damaging agentand is subsequently contacted with a p53 protein or gene.
 14. The methodof claim 1, wherein said cell is simultaneously contacted with a p53protein or gene and a DNA damaging agent.
 15. The method of claim 1,wherein said cell is contacted with a first composition comprising a p53protein or gene and a second composition comprising a DNA damagingagent.
 16. The method of claim 15, wherein said first or secondcomposition is dispersed in a pharmacologically acceptable formulation.17. The method of claim 1, wherein said cell is contacted with a singlecomposition comprising a p53 protein or gene in combination with a DNAdamaging agent.
 18. The method of claim 17, wherein said composition isdispersed in a pharmacologically acceptable formulation.
 19. The methodof claim 17, wherein said cell is contacted with a single compositioncomprising a recombinant vector that expresses p53 in said cell incombination with a DNA damaging agent.
 20. The method of claim 19,wherein said cell is contacted with a single composition comprising arecombinant adenovirus containing a recombinant vector that expressesp53 in said cell in combination with a DNA damaging agent.
 21. Themethod of claim 1, wherein said cell is a human cell.
 22. The method ofclaim 1, wherein said cell is a malignant cell.
 23. The method of claim22, wherein said cell is a lung cancer cell.
 24. The method of claim 22,wherein said cell is a breast cancer cell.
 25. The method of claim 22,wherein said cell has a mutation in a p53 gene.
 26. The method of claim1, wherein said cell is located within an animal and said p53 protein orgene and DNA damaging agent are administered to the animal in apharmacologically acceptable form.
 27. A method of treating cancer,comprising administering to an animal with cancer a therapeuticallyeffective combination of a p53 protein or gene and a DNA damaging agent.28. The method of claim 27, comprising injecting into a tumor site atherapeutically effective amount of a pharmaceutical compositioncomprising a recombinant adenovirus containing a recombinant vector thatexpresses p53 in the tumor cell, and contacting the tumor with a DNAdamaging agent.
 29. The method of claim 28, wherein the tumor iscontacted with a DNA damaging agent by irradiating the tumor site withX-ray radiation, UV-irradiation, T-irradiation or microwaves.
 30. Themethod of claim 28, wherein the tumor is contacted with a DNA damagingagent by administering to the animal a therapeutically effective amountof a pharmaceutical composition comprising a DNA damaging compound. 31.The method of claim 28, wherein the DNA damaging compound is cisplatin.32. A composition comprising a p53 protein or gene in combination with aDNA damaging agent.
 33. The composition of claim 32, comprising a p53protein or gene in combination with adriamycin, 5-fluorouracil,etoposide, camptothecin, actimomycin-D, mitomycin C, or cisplatin. 34.The composition of claim 33, comprising a p53 protein or gene incombination with cisplatin.
 35. The composition of claim 32, comprisinga recombinant vector that expresses a p53 protein in an animal cell incombination with a DNA damaging agent.
 36. The composition of claim 35,wherein said recombinant vector is a naked DNA plasmid, a plasmid withina liposome, a retroviral vector, an AAV vector, or a recombinantadenoviral vector.
 37. The composition of claim 36, wherein saidrecombinant vector is a recombinant adenoviral vector.
 38. Thecomposition of claim 37, wherein said recombinant vector is arecombinant adenoviral vector is present within a recombinant adenovirusparticle.
 39. The composition of claim 32, comprising a recombinantadenoviral vector present within a recombinant adenovirus particle incombination with cisplatin.
 40. The composition of claim 32, dispersedin a pharmacologically acceptable formulation.
 41. The composition ofclaim 40, formulated for intralesional administration.
 42. A therapeutickit comprising, in suitable container means, a pharmaceuticalformulation of a recombinant vector that expresses a p53 protein in ananimal cell and a pharmaceutical formulation of a DNA damaging agent.43. The kit of claim 42, wherein said recombinant vector and said DNAdamaging agent are present within a single container means.
 44. The kitof claim 42, wherein said recombinant vector and said DNA damaging agentare present within distinct container means.
 45. The kit of claim 42,comprising a pharmaceutical formulation of a recombinant adenovirusincluding a recombinant vector that expresses a p53 protein in an animalcell and a pharmaceutical formulation of cisplatin.